A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes.

AIMS: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. METHODS AND RESULTS: We optimized the conditions for transformation using a plasmid pUCYIT356-1-Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. CONCLUSIONS: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.

Complete DNA sequence and comparative analysis of the 50-kilobase virulence plasmid of Salmonella enterica serovar Choleraesuis.

The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidal Salmonella. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef (plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coli O157:H7 and the enteropathogenic E. coli (EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of Salmonella serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.

Construction of mutants of Sphingomonas paucimobilis defective in terminal mannose in the glycosphingolipid.

Mutants of Sphingomonaspaucimobilis defective in a part of the carbohydrate moiety of the glycosphingolipid (GSL) were constructed by transposon (Tn5)-insertional mutagenesis. Defective mutants were selected by ELISA using the antibody recognizing the tetrasaccharide-type GSL (GSL-4A) of S. paucimobilis. Eight defective mutants were selected from about 8,000 kanamycin-resistant strains, and seven of them were found to lack the terminal mannose of GSL-4A. The chemical structure of the mutant GSL was investigated, and proved that the rest of the structure was not changed by the mutation.

Constitutively expressed phoP inhibits mouse-virulence of Salmonella typhimurium in an Spv-dependent manner.

In Salmonella typhimurium, the transcription of several virulence genes including spvB is regulated by the PhoP/PhoQ regulatory system. To further examine the relationship between the PhoP/PhoQ and Spv systems for virulence in mice, we examined a non-polar phoP mutation combined with different virulence plasmid genotypes for effects on virulence of S. typhimurium in the mouse model. PhoP-/Spv+ and PhoP-/Spv- mutants were not detectably recovered from the spleens of subcutaneously or orally inoculated mice. The phoP gene constitutively expressed from the lacZ promoter of a low copy number vector (phoP(C)) only partially complemented the non-polar phoP mutation for mouse-virulence in both the Spv+ and Spv- backgrounds; both PhoP(C) strains exhibited virulence equal only to a PhoP+/Spv- strain. Interestingly, in a PhoP+ background, the phoP(C) gene reduced splenic infection of the Spv+ but not Spv- salmonellae after subcutaneous or oral inoculation compared with the PhoP+ parents. Additionally, the phoP(C) gene in an Spv+ background reduced the net growth of salmonellae in macrophages in vitro; phoP(C) in an Spv- background was without effect. These data suggest that the constitutive expression of the phoP gene attenuates the virulence of S. typhimurium in mice in an Spv-dependent manner.

Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus.

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.

Cloning of the gene coding for Staphylococcus hyicus exfoliative toxin B and its expression in Escherichia coli.

The Staphylococcus hyicus exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in Escherichia coli. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.

TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.

Formation of ring-shaped structures on erythrocyte membranes after treatment with botulinolysin, a thiol-activated hemolysin from Clostridium botulinum.

Damage to erythrocyte membranes by botulinolysin (BLY) was studied by electron microscopy, which revealed ring-shaped structures with inner diameters and widths of approximately 32 and 6.7 nm, respectively. BLY bound to membranes at 0 degrees C, but subsequent treatment with glutaraldehyde prevented ring formation during further incubation at 37 degrees C. Zn2+ ions inhibited ring formation but not binding of BLY to membranes.

Comparison of characterization among Bordetella calmodulin-like protein, bovine brain calmodulin and Escherichia coli acyl-carrier protein.

We previously reported that Bordetella calmodulin-like protein (CLP), like bovine brain calmodulin (CaM), enhances adenylate cyclase and phosphodiesterase activities. In this communication, antigenic and biochemical characters were compared between CLP and CaM. It was found that anti-CLP and anti-CaM sera obtained reacted with CaM and CLP, respectively. The amino acid composition of CLP was similar to CaM. The similarity was also found in an Escherichia coli acyl-carrier protein (ACP), a Ca(2+)-binding protein. Though the amino acid sequence of N-terminus region of CLP has no significant homology with CaM, ACP has highly homology in its N-terminus similar to CaM. These results suggest that CLP might act as a Ca(2+)-binding protein, and/or an ACP during the activation of adenylate cyclase and phosphodiesterase.

Inhibitory effect of acyl-CoA and acyl-carnitine compounds on the ischemia-inducing activity of Bordetella heat-labile toxin in guinea pig skin.

The ability of Bordetella heat-labile toxin (HLT) to induce ischemic lesions after intracutaneous injection to guinea pig skin was lost following incubation at 37 degrees C with long-chain saturated acyl-CoA and acyl-carnitine compounds. Short-chain unsaturated acyl-CoA compounds, however, were less potent in inhibiting the induction of HLT activity. Long-chain saturated acyl-CoA and acyl-carnitine compounds, potently inhibited the induction of this activity. On incubation with HLT at 0 degrees C, a long-chain saturated acyl-CoA compound, palmitoyl-CoA, did not inhibit HLT activity. When first mixed with bovine serum albumin or dimethyl-beta-cyclodextrin, palmitoyl-CoA lost the ability to inhibit HLT activity. Binding of 14C-palmitoyl-CoA to HLT was measured by Scatchard analysis. The Bmax values of HLT (2.75 mol/mol of protein) were higher than that of acyl-CoA-binding protein from bovine liver (0.95 mol/mol of protein). Neither acyl-CoA hydrolase nor acyl-CoA ligase was detected in the HLT preparation. These results suggest that the acyl-CoA and acyl-carnitine compounds bind directly to HLT and produce a critical change in conformation required for HLT activity.

Induction of TNF-alpha mRNA in murine macrophages by virulent and avirulent strains of Salmonella choleraesuis serovar typhimurium and serovar Choleraesuis.

TNF-alpha mRNA induction in murine macrophages by virulent and avirulent Salmonella strains was measured in vitro by RT-PCR method. Virulence plasmid-cured strains of S. choleraesuis serovar Typhimurium and serovar Choleraesuis, and rpoS-defective mutant of S. choleraesuis serovar Typhimurium induced significantly higher level of TNF-alpha mRNA than their parent (virulent) strains in macrophages of C3H/HeN mice. When macrophages of LPS-low responder (C3H/HeJ) mice were used, the difference of the induction level was not observed, indicating that LPS was involved in the enhanced level of TNF-alpha mRNA induction by avirulent Salmonella strains. LPSs from virulent and avirulent strains were analysed, but no difference was found for cytokine-inducing activity, and chemical properties. Those results suggested that avirulent Salmonella strains were damaged more easily, and released more LPS in macrophages to enhance TNF-alpha induction.

Electron microscopic evaluation of a two-step theory of pore formation by streptolysin O.

The formation of pores by streptolysin O (SLO) was analyzed in erythrocyte membranes and liposomes by immunoelectron microscopy and electron spectroscopic imaging. The binding of SLO molecules to membranes was temperature independent, while the polymerization of SLO molecules was temperature dependent. Our results also suggest that proteins in erythrocyte membranes are not involved in the formation of SLO rings.

Production of antibody against calmodulin which is a protein with poor antigenicity.

Calmodulin (CaM), a Ca(2+)-binding protein, is a multifunctional modulator mediating the calcium regulation of a large number of intracellular enzyme systems. Because of its highly conserved structure, it has been difficult to produce anti-CaM antibody. We describe here a simplified method for production of this antibody. BALB/c mice were immunized with a polymerized CaM, and the serum was prepared and tested for the presence of anti-CaM antibody by immunoblot analysis. The serum obtained from the immunized mice was shown to contain anti-CaM antibody. Immunoblot analysis proved that a 500-fold diluted serum was able to recognize both types of CaM with a polymerized form and monomer form. The method developed in this study may be applicable to a wide range of proteins for enhancement of antigenicity.

Failure of smooth muscle cells treated with collagenase to respond to Bordetella heat-labile toxin.

HLT, bordetella heat-labile toxin, caused a release of radioactivity from cultured smooth muscle cells (SMC) previously labeled with [14C]arachidonic acid, and from membrane preparations from the labeled SMC. HLT, however, failed to induce release from primary SMC prepared by treatment with an enzyme solution containing collagenase and elastase. When cultured SMC were treated with the enzyme solution, their susceptibility to HLT was reduced more significantly than that of untreated cells. SMC also lost their susceptibility, after treatment with collagenase alone, but not with elastase, in a dose-dependent manner. These data suggest that collagen on the SMC surface might play an important role in HLT activity.

Construction and evaluation of a virG thyA double mutant of Shigella flexneri 2a as a candidate live-attenuated oral vaccine.

A virG thyA double mutant of Shigella flexneri 2a was constructed as a candidate live-attenuated oral vaccine. In the keratoconjunctivitis model it did not provoke any adverse reaction by itself on guinea pigs' eyes and completely protected them from provoking keratoconjunctivitis. When (2.7-4.8) x 10(10) of the vaccine was inoculated intragastrically after 1 day fasting in cynomolgus monkeys three times at weekly intervals, a watery stool was observed at 40% as a side-effect. Upon intragastric challenge after 1 day fasting with 7.5 x 10(9) of the virulent strain four weeks after the last vaccination, a statistically significant difference was obtained in the mortality rate but not in the morbidity rate between the vaccine and the control group, although the clinical findings were less severe in the vaccine group than in the control group. These results together with the histopathological and immunological findings indicate that the vaccine deserve further detailed studies.

Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine.

Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response. The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT. Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype. These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza.

Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis.

The expression regulation of spvR, a regulatory gene on the virulence plasmid (pKDSC50) of Salmonella choleraesuis serovar Choleraesuis, was investigated by spvR-lacZ translational fusion. The spvR gene was found to be positively regulated by its own product, the SpvR protein, and this unusual positive autoregulation was repressed by the products of spvA and spvB, virulence-associated genes present downstream from the spvR gene. Amino acid sequence analysis revealed that the amino-terminal region of SpvB had homology with the CatM repressor protein of Acinetobacter calcoaceticus, which belongs to the MetR/LysR protein family. On the other hand, the sigma factor RpoS was required for expression of the spvR gene in the stationary phase of bacterial growth. The SpvR protein was also necessary for self-activation, suggesting that an RNA polymerase holoenzyme containing RpoS requires SpvR protein in order to recognize the spvR promoter.

Virulence region of plasmid pNL2001 of Salmonella enteritidis.

The virulence region of the Salmonella enteritidis 55 kb plasmid pNL2001 was identified by Tn1-insertion mutagenesis, DNA hybridization studies, and Western blot analysis of proteins encoded in the virulence region of the plasmid. DNA hybridization studies showed that the pNL2001 plasmid contained a 6.4 kb SalI-EcoRI fragment homologous to the 6.4 kb SalI-EcoRI Salmonella plasmid virulence (spv) region of the S. choleraesuis 50 kb plasmid (pKDSC50). One of the 247 Tn1-insertion mutants of S. enteritidis, designated strain TA19, showed a reduced mouse lethality, and the Tn1-insertion of strain TA19 was located within this homologous 6.4 kb region, suggesting that the 6.4 kb SalI-EcoRI fragment of pNL2001 contained the spv region. Two contiguous SalI-EcoRI fragment were cloned into the expression vectors. By the 6.4 kb SalI-EcoRI fragment were cloned into the expression vectors. By Western blot analysis using four Spv peptide antisera, each specific for individual proteins encoded in the spvR, spvA, spvB and spvC genes of pKDSC50, four proteins encoded in the 6.4 kb SalI-EcoRI fragment of pNL2001 were identified. Protein SpvR with an apparent molecular mass of 32 kDa was produced from the 2.3 kb SalI-EcoRI fragment, and protein SpvA, SpvB and SpvC with apparent molecular masses of 32, 70 and 29 kDa, respectively, were produced from the 4.1 kb EcoRI-EcoRI fragment. From the 4.1 kb EcoRI::Tn1 fragment of the TA19 plasmid, proteins SpvA and SpvB were expressed, but not SpvC. It was therefore suggested that the spvC gene may contribute to the expression of virulence of S. enteritidis. Furthermore, the nucleotide sequence of the 6.4 kb SalI-EcoRI fragment encoding these four proteins was determined. Four open reading frames which encoded the four proteins with deduced molecular masses of 33,906, 28,200, 65,349 and 27,646 Da were detected. Deduced amino acid sequences of each protein showed a high degree of identity to corresponding sequences in the virulence region of S. choleraesuis, S. dublin and S. typhimurium virulence plasmids. Therefore, we confirmed that the virulence plasmids of Salmonellae including S. enteritidis share the highly conserved region responsible for virulence.

Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine.

Cholera toxin B subunit (CTB) and Escherichia coli heat-labile toxin (LTB) (2 micrograms), each supplemented with a trace amount of cholera toxin (CT) (0.02-20 ng), were examined for the adjuvant effect on antibody (Ab) response against influenza inactivated HA (haemagglutinin) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation of the vaccine (1.5 micrograms) and the CT-containing CTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The primary inoculation of the vaccine with CTB alone did not induce either anti-HA IgA or IgG Ab response, or haemagglutination-inhibition Ab responses in the serum. The vaccine with less than 2 ng of CT also failed to induce Ab response. On the other hand, the vaccine with CT-containing CTB induced a high Ab response, which increased depending on the CT dose. Moreover, the second vaccine induced a response more than ten times higher than the primary one and the response increased depending on the CT dose. Similar enhancement was found in the local anti-HA IgA Ab response in the nasal wash. Such synergistic effects were observed also between LTB and CT. The amount of Ab produced by the synergism was considered to be enough to protect against virus infection. These results suggest that CTB (or LTB) containing a trace amount of CT (about 0.1%) can be used practically as a potent adjuvant for nasal vaccination of humans against influenza.

Purification and characterization of Bordetella calmodulin-like protein.

Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca(2+)-dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca2+ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis.