Genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China.

In this report, we present the complete genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China. The sequencing was conducted using a hybrid assembly methodology that combined Illumina short reads and PacBio long reads. This approach provides a high-quality representative sequence for the strains mentioned above.

Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

Genome Sequence and Phenotypic Analysis of a Protein Lysis-Negative, Attenuated Anthrax Vaccine Strain.

Bacillus anthracis is a Gram-positive bacterium that causes the zoonotic disease anthrax. Here, we studied the characteristic phenotype and virulence attenuation of the putative No. II vaccine strain, PNO2, which was reportedly introduced from the Pasteur Institute in 1934. Characterization of the strain showed that, compared with the control strain, A16Q1, the attenuated PNO2 (PNO2D1) was phospholipase-positive, with impaired protein hydrolysis and significantly reduced sporulation. Additionally, PNO2D1 significantly extended the survival times of anthrax-challenged mice. An evolutionary tree analysis revealed that PNO2D1 was not a Pasteur strain but was more closely related to a Tsiankovskii strain. A database comparison revealed a seven-base insertion mutation in the nprR gene. Although it did not block nprR transcription, the insertion mutation resulted in the premature termination of protein translation. nprR deletion of A16Q1 resulted in a nonproteolytic phenotype that could not sporulate. The database comparison revealed that the abs gene is also prone to mutation, and the abs promoter activity was much lower in PNO2D1 than in A16Q1. Low abs expression may be an important reason for the decreased virulence of PNO2D1.

Bioinformatic analysis of structures and encoding genes of Escherichia coli surface polysaccharides sheds light on the heterologous biosynthesis of glycans.

BACKGROUND: Surface polysaccharides (SPs), such as lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen), play a key role in the pathogenicity of Escherichia coli (E. coli). Gene cluster for polysaccharide antigen biosynthesis encodes various glycosyltransferases (GTs), which drive the process of SP synthesis and determine the serotype. RESULTS: In this study, a total of 7,741 E. coli genomic sequences were chosen for systemic data mining. The monosaccharides in both O and K antigens were dominated by D-hexopyranose, and the SPs in 70-80% of the strains consisted of only the five most common hexoses (or some of them). The linkages between the two monosaccharides were mostly α-1,3 (23.15%) and ß-1,3 (20.49%) bonds. Uridine diphosphate activated more than 50% of monosaccharides for glycosyltransferase reactions. These results suggest that the most common pathways could be integrated into chassis cells to promote glycan biosynthesis. We constructed a database (EcoSP, http://ecosp.dmicrobe.cn/ ) for browse this information, such as monosaccharide synthesis pathways. It can also be used for serotype analysis and GT annotation of known or novel E. coli sequences, thus facilitating the diagnosis and typing. CONCLUSIONS: Summarizing and analyzing the properties of these polysaccharide antigens and GTs are of great significance for designing glycan-based vaccines and the synthetic glycobiology.

Genome Sequence of Acinetobacter baumannii Strain SHOU-Ab01, Isolated from Chinese Giant Salamander (Andrias davidianus) Liver.

This report describes the complete genome sequence of Acinetobacter baumannii strain SHOU-Ab01, which was isolated from the liver of a Chinese giant salamander (Andrias davidianus). SHOU-Ab01 belonged to sequence type 40 (ST40), and its genome contained a circular chromosome (size, 3,891,862 bp) and two circular plasmids (sizes, 8,571 bp and 5,870 bp).

Rapid Identification of Bacillus anthracis In Silico and On-Site Using Novel Single-Nucleotide Polymorphisms.

Bacillus anthracis is a spore-forming bacterium that causes life-threatening infections in animals and humans and has been used as a bioterror agent. Rapid and reliable detection and identification of B. anthracis are of primary interest for both medical and biological threat-surveillance purposes. Few chromosomal sequences provide enough polymorphisms to clearly distinguish B. anthracis from closely related species. We analyzed 18 loci of the chromosome of B. anthracis and discovered eight novel single-nucleotide polymorphism (SNP) sites that can be used for the specific identification of B. anthracis. Using these SNP sites, we developed software-named AGILE V1.1 (anthracis genome-based identification with high-fidelity E-probe)-for easy, user-friendly identification of B. anthracis from whole-genome sequences. We also developed a recombinase polymerase amplification-Cas12a-based method that uses nucleic acid extracts for the specific, rapid, in-the-field identification of B. anthracis based on these SNPs. Via this method and B. anthracis-specific CRISPR RNAs for the target CR5_2, CR5_1, and Ba813 SNPs, we clearly detected 5 aM genomic DNA. This study provides two simple and reliable methods suitable for use in local hospitals and public health programs for the detection of B. anthracis. IMPORTANCE Bacillus anthracis is the etiologic agent of anthrax, a fatal disease and a potential biothreat. A specific, accurate, and rapid method is urgently required for the identification of B. anthracis. We demonstrate the potential of using eight novel SNPs for the rapid and accurate detection of B. anthracis via in silico and laboratory-based testing methods. Our findings have important implications for public health responses to disease outbreaks and bioterrorism threats.

A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis.

As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

Feeding preference of insect larvae to waste electrical and electronic equipment plastics.

Waste electrical and electronic equipment (WEEE) plastics not only pollute the environment, but are challenging to treat in an environmentally friendly manner. Biodegradation by insect larvae is potentially an eco-friendly method to treat WEEE plastics, but information about the feeding preference of insect larvae to WEEE plastics is lacking. In this study, a total of nine WEEE and pristine plastics were chosen to feed larvae of the following two insect species, i.e. Galleria mellonella and Tenebrio molitor. G. mellonella larvae significantly favor corresponding pristine plastics compared to two types of WEEE plastics, waste rigid polyurethane (RPU) and waste polystyrene (PS). One possible explanation is the increased chlorine or metals in the WEEE plastics measured using X-ray fluorescence spectrometer analysis. Scanning electron microscopy and Fourier transform infrared spectroscopy show that the destruction of physical structures and changes in surface functional groups were found in the two types of WEEE plastics in the larval frass, implying that the larvae partly biodegraded the plastics. Meanwhile, the powdered waste high impact polystyrene plastics (WHIPS) were ingested, but not the lumpy ones, indicating that the consumption by G. mellonella larvae is improved by the WHIPS physical modification. In addition, G. mellonella larvae presented the following decreasing preference for pristine plastics under individual-plastic-fed mode: RPU > phenol-formaldehyde resin > polyethylene (PE) > polypropylene > PS ≈ polyvinyl chloride; this is possibly due to differences in physical properties and chemical structures of the plastics; feeding preference of the larvae under multiple-plastics-fed mode is relatively consistent to that under individual-plastic-fed mode. Interestingly, the consumption by G. mellonella larvae of PE is higher than that of PS, while T. molitor larvae showed the opposite trend, implying that insect larvae have different plastics preference. The findings provide insights into biodegradation of WEEE plastics by insect larvae.

Obtaining Specific Sequence Tags for Yersinia pestis and Visually Detecting Them Using the CRISPR-Cas12a System.

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR-fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains.

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some "scissors plasmids" via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96-100%), rather than the non-replication initiation region (88-92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

Biosynthesis of Self-Assembled Proteinaceous Nanoparticles for Vaccination.

Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi-biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology-only processes like complex sequential post-translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano-B5 platform for in vivo production of fully protein-based, self-assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self-assembly capacities of pentamer domains from the bacterial AB5 toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano-B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

SpoVG is Necessary for Sporulation in Bacillus anthracis.

The Bacillus anthracis spore constitutes the infectious form of the bacterium, and sporulation is an important process in the organism's life cycle. Herein, we show that disruption of SpoVG resulted in defective B. anthracis sporulation. Confocal microscopy demonstrated that a ΔspoVG mutant could not form an asymmetric septum, the first morphological change observed during sporulation. Moreover, levels of spoIIE mRNA were reduced in the spoVG mutant, as demonstrated using ß-galactosidase activity assays. The effects on sporulation of the ΔspoVG mutation differed in B. anthracis from those in B. subtilis because of the redundant functions of SpoVG and SpoIIB in B. subtilis. SpoVG is highly conserved between B. anthracis and B. subtilis. Conversely, BA4688 (the protein tentatively assigned as SpoIIB in B. anthracis) and B. subtilis SpoIIB (SpoIIBBs) share only 27.9% sequence identity. On complementation of the B. anthracis ΔspoVG strain with spoIIBBs, the resulting strain pBspoIIBBs/ΔspoVG could not form resistant spores, but partially completed the prespore engulfment stage. In agreement with this finding, mRNA levels of the prespore engulfment gene spoIIM were significantly increased in strain pBspoIIBBs/ΔspoVG compared with the ΔspoVG strain. Transcription of the coat development gene cotE was similar in the pBspoIIBBs/ΔspoVG and ΔspoVG strains. Thus, unlike in B. subtilis, SpoVG appears to be required for sporulation in B. anthracis, which provides further insight into the sporulation mechanisms of this pathogen.

Genotyping and population diversity of Bacillus anthracis in China based on MLVA and canSNP analysis.

In pastoral parts of China, anthrax still presents a major risk to livestock and threatens the health of local human populations. Currently, whole-genome-based molecular markers, such as single-nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs), are the most effective tools for genotyping Bacillus anthracis. In this study, 191 isolates were selected to assess the diversity of B. anthracis in China. Five isolates were confirmed not to be B. anthracis by clustered regularly interspaced short palindromic repeat analysis, while the remaining 186 isolates were typed using canonical SNP (canSNP) and VNTR analyses. Five sublineages/subgroups, A.Br.001/002, A.Br.Vollum, A.Br.Aust.94, A.Br.Ames, and A.Br.008/009, were detected based on 13 canSNP sites. The 186 isolates were further assigned 114 sequence types based on 27 VNTR loci, with major branches correlating with the canSNP analysis. We then used a simplified multiple-locus variable number tandem repeat analysis (MLVA) protocol (MLVAmin) based on eight high-resolution VNTR sites to analyze the Chinese isolates, with the resulting phylogeny again agreeing with the canSNP analysis. We also developed two schemes, MLVAc and MLVAp, using various numbers of VNTRs to analyze different canSNP sublineages to increase the typing resolution of the canSNP protocol. The results showed a highly imbalanced geographical distribution of the B. anthracis population, with four different sublineages observed in Xinjiang Province, while only one sublineage, A.Br.001/002, was found in the other six provinces, except for three A.Br.Ames strains isolated from Inner Mongolia. Based on the MLVA and canSNP analysis, the spread of B. anthracis appears to have occurred from west to east via three independent routes.

Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System.

Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in B. anthracis as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In B. cereus, we successfully introduced precise point mutations into plcR, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to B. anthracis, which is a natural plcR mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the Bacillus cereus group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant B. cereus.

Disruption of SpoIIID decreases sporulation, increases extracellular proteolytic activity and virulence in Bacillus anthracis.

Endospores are important for maintenance of the B. anthracis lifecycle and necessary for its effective spread between hosts. Our experiments with B. anthracis showed that disruption of SpoIIID results in a spore formation defect, as determined by heat resistance assays and microscopic assessment. We further found complete engulfment by the ΔspoIIID mutant strain by membrane morphology staining but no synthesis of the clarity coat and exosporium by transmission electron microscopy. Reduced transcription and expression of small acid-soluble spore protein(sasP-2) and the spore development associated genes (σK, gerE and cotE) in the mother cell were found in the ΔspoIIID strain, suggesting that the spore formation defect in B. anthracis A16R is related to decreased transcription and expression of these genes. Extracellular protease and virulence enhancement in the ΔspoIIID strain may be related to the elevation of metalloproteinases (TasA and Camelysin) levels. Our findings pave the way for further research on the regulation network of sporulation, survival and virulence in these two morphological forms of B. anthracis.

Genome sequence of Bacillus anthracis typing phage AP631.

AP631, a virulent bacteriophage of Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.

Design and production of conjugate vaccines against S. Paratyphi A using an O-linked glycosylation system in vivo.

Enteric fever, mainly caused by Salmonella enterica serovar Paratyphi A, remains a common and serious infectious disease worldwide. As yet, there are no licensed vaccines against S. Paratyphi A. Biosynthesis of conjugate vaccines has become a promising approach against bacterial infection. However, the popular biosynthetic strategy using N-linked glycosylation systems does not recognize the specialized O-polysaccharide structure of S. Paratyphi A. Here, we describe an O-linked glycosylation approach, the only currently available glycosylation system suitable for an S. Paratyphi A conjugate vaccine. We successfully generated a recombinant S. Paratyphi A strain with a longer O-polysaccharide chain and transformed the O-linked glycosylation system into the strain. Thus, we avoided the need for construction of an O-polysaccharide expression vector. In vivo assays indicated that this conjugate vaccine could evoke IgG1 antibody to O-antigen of S. Paratyphi A strain CMCC 50973 and elicit bactericidal activity against S. Paratyphi A strain CMCC 50973 and five other epidemic strains. Furthermore, we replaced the peptides after the glycosylation site (Ser) with an antigenic peptide (P2). The results showed that the anti-lipopolysaccharide antibody titer, bactericidal activity of serum, and protective effect during animal challenge could be improved, indicating a potential strategy for further vaccine design. Our system provides an easier and more economical method for the production of S. Paratyphi A conjugate vaccines. Modification of the glycosylation site sequon provides a potential approach for the development of next-generation "precise conjugate vaccines."

Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis.

To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased rate of allelic exchange. Golden Gate cloning is a molecular cloning strategy allowing researchers to simultaneously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction enzymes and T4 DNA ligase in the same reaction system. Here, a B. anthracis S-layer protein EA1 allelic exchange vector was successfully constructed using the Golden Gate method. No new restriction sites were introduced into this knockout vector, and seamless assembly of the DNA fragments was achieved. To elevate the efficiency of homologous recombination between the allelic exchange vector and chromosomal DNA, we introduced an I-SceI site into the allelic exchange vector. The eag gene was successfully knocked out in B. anthracis using this vector. Simultaneously, the allelic exchange vector construction method was developed into a system for generating B. anthracis allelic exchange vectors. To verify the effectiveness of this system, some other allelic exchange vectors were constructed and gene replacements were performed in B. anthracis. It is speculated that this gene knockout vector construction system and high-efficiency targeted gene replacement using I-SceI endonuclease can be applied to other Bacillus spp.

An H-NS Family Protein, Sfh, Regulates Acid Resistance by Inhibition of Glutamate Decarboxylase Expression in Shigella flexneri 2457T.

The glutamate-dependent acid-resistance system is the most effective acid tolerance pathway in Shigella, allowing survival in extremely acidic environments. However, the regulation of this system in Shigella remains elusive. In the current study, we identified significant differences in the levels of glutamate decarboxylase between three Shigella flexneri strains with different levels of acid resistance using blue native-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF)/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the degree of acid resistance and the levels of GadA/B were significantly lower in strain 2457T compared with two other S. flexneri strains. It has been reported that plasmid pSf-R27 is expressed in strain 2457T but not in the other 142 sequenced S. flexneri isolates. pSf-R27 encodes protein Sfh, which belongs to a family of histone-like nucleoid-structuring (H-NS) proteins that participate in the transcriptional control of glutamate-dependent acid resistance, implicating pSf-R27 in the lower acid resistance of strain 2457T. Transformation of pSf-R27 or sfh alone into strain 301 resulted in decreased expression of GadA/B in the recombinant strains. Thus, we confirmed that H-NS family protein Sfh, bound to the gadA/B regulatory region and regulates the expression of glutamate decarboxylase at the transcriptional level. We also examined the acid tolerance of the wild-type and recombinant strains using flow cytometry and determined that the acid tolerance of S. flexneri is closely related to the expression of GadA/B. These findings further our understanding of the acid tolerance of S. flexneri, especially via the glutamate-dependent pathway.

Role of the virulence plasmid in acid resistance of Shigella flexneri.

Virulence plasmid (VP) acquisition was a key step in the evolution of Shigella from a non-pathogenic Escherichia coli ancestor to a pathogenic genus. In addition, the co-evolution and co-ordination of chromosomes and VPs was also a very important step in the evolutionary process. To investigate the cross-talk between VPs and bacterial chromosomes, we analyzed the expression profiles of protein complexes and protein monomers in three wild-type Shigella flexneri strains and their corresponding VP deletion mutants. A non-pathogenic wild-type E. coli strain and mutant E. coli strains harboring three Shigella VPs were also analyzed. Comparisons showed that the expression of chromosome-encoded proteins GadA/B and AtpA/D, which are associated with intracellular proton flow and pH tuning of bacterial cells, was significantly altered following acquisition or deletion of the VP. The acid tolerance of the above strains was also compared, and the results confirmed that the presence of the VP reduced the bacterial survival rate in extremely acidic environments, such as that in the host stomach. These results further our understanding of the evolution from non-pathogenic E. coli to Shigella, and highlight the importance of co-ordination between heterologous genes and the host chromosome in the evolution of bacterial species.