Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

Detection of Horizontal Gene Transfer by Natural Transformation in Native and Introduced Species of Bacteria in Marine and Synthetic Sediments

Both naturally occurring marine sediments and artificial sediments were used as supports for natural transformation of marine bacteria. While transformation of Pseudomonas stutzeri ZoBell suspended in artificial seawater was not detected when recipient cells and rifampin resistance DNA were loaded onto sterile sediment columns, transformation could be detected at frequencies 4 to 20 times that of spontaneous resistance when recipient cells and rifampin resistance DNA were loaded onto sterile sediment columns. Treatment of these columns with DNase I reduced transformation frequencies to levels comparable to those of spontaneous-resistance frequencies. Sediments with higher organic contents supported higher frequencies of transformation than did those with lower amounts of organic matter. Transformation was also detected when recipient cells and DNA were loaded on columns prepared from nonsterile sediments, although the frequencies of transformation were lower than when sterile sediments were used. Finally, nonsterilized sediments that were not supplemented with laboratory strains did not support detectable levels of transformation in sediment columns, but when these same sediments were transferred to filters and placed on complex media, transformation was detected at a frequency three times that for spontaneous resistance. This transformation frequency was partially reduced to levels near that for spontaneous resistance by the addition of DNase I to sediment filters. These results indicate that marine sediments facilitate the uptake and expression of exogenous DNA by transformable marine bacteria and that sediments are a more likely niche for natural transformation than the water column in the marine environment.     

Reaction of the hematopoietic system in mice to the administration of virulent and avirulent strains of Shigella flexneri 2A

The influence of live bacteria and antigenic preparations of virulent and avirulent stains of Shigella flexneri 2a on the endogenous splenic colony formation in murine hematopoietic cells has been studied. The different stimulating activity of live microbial cells of virulent and avirulent strains Shigella flexneri 2a and their antigenic preparations on the endogenous colony formation has been shown. The effect observed depends on the preparation doses and time of their administration before irradiation of the animals. The stimulating influence on the development of hemopoiesis endogenous foci may be conditioned by the action of heat-labile products of the live microbial cells and closely correlates with the virulence of strains studied.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

Transformation of Haemophilus influenzae by plasmid RSF0885.

Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximum, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Behavior of Coliphage Lambda in Shigella flexneri 2a

The insensitivity of wild-type Shigella flexneri 2a to coliphage lambda is a consequence of its native genetic defect in the malA gene cluster. The "smooth" S. flexneri 2a lipopolysaccharide layer affects the efficient adsorption of lambda. Derivatives, capable of serving as functional hosts for lambda, were obtained by repairing the malA lesion, enabling the expression of the malB-lambdarcp region of S. flexneri. Introduction of a mutation into S. flexneri causing a "rough" lipopolysaccharide character resulted in more efficient adsorption of lambda. Such S. flexneri hosts can be stably lysogenized and upon induction yield gal(+)-transducing lysates. Lambda propagated on a malA(+) rough S. flexneri host was restricted by Escherichia coli K-12 and E. coli B, but not by E. coli C. This S. flexneri host did not restrict lambda grown on these E. coli strains.     

Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence.

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.     

鲍氏志架氏菌的一个新血清型

Two strains which belong to the same serotype of Shigella were isolated from the bloody-pus stool of two patients (in 1986) and is reported in this paper. The results were identical both showing agglutination in low titer with serotype 8 of S. dysenteriae and serotype 4 of S. boydii when the two strains were checked well with all kinds of diagnostic antisera and vice versa, ie the antisera produced by the two strains were also checked well with sera prepared with the representative strains of all Shigella spp. No cross agglutination with O6, O7, and O150 of E. coli were found. Consequently, It appears to be a new serotype of Shigella. These two strains possess the ability of causing keratitis in guinea-pigs as well as invading epithelial cells, the DNA of both strains in agarose-electrophoresis showed a large plasmid, indicating that they are virulent strains possessing invasive ability. It was concluded that these two strains belonged to Shigella boydii as they fermented mannitol and non-related antigenically with Shigella flexneri. Since serotype 1-18 of S. boydii have been reported recently, we propose that this new serotype should be serotype 19 of Shigella boydii.     

Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication

Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

Correlation between Congo red binding and contact haemolysin production in Shigella species

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.     

Construction of a multivalent vaccine strain of Shigella flexneri and evaluation of serotype-specific immunity

Shigella flexneri causes more fatalities by shigellosis than any other Shigella species. There are 13 different serotypes of S. flexneri and their distribution varies between endemic geographical regions. The immune response against S. flexneri is serotype-specific, so current immunization strategies have required the administration of multiple vaccine strains to provide protection against multiple serotypes. In this study, we report the construction of a multivalent S. flexneri vaccine strain, SFL1425, expressing the O-antigen structure specific for serotypes 2a and 5a. This combination of type antigens has not previously been reported for S. flexneri. The multivalent vaccine strain, SFL1425 was able to induce a specific immune response against both serotypes 2a and 5a in a mouse pulmonary model.