Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Bacterial rhomboid proteases mediate quality control of orphan membrane proteins.

Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.

An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei.

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

A comparative study of the evolution of cellobiose utilization in Escherichia coli and Shigella sonnei.

The chb operon of Escherichia coli is involved in the utilization of chitooligosaccharides. While acquisition of two classes of mutations leading to altered regulation of the chb operon is necessary to confer the ability to utilize the glucose disaccharide cellobiose to wild-type strains of E. coli, in the closely related organism Shigella sonnei, Cel+ mutants arise relatively faster, requiring only a single mutational event. In Type I mutants, the insertion of IS600 at -21 leads to ChbR regulator-independent, constitutive expression of the operon. In Type II mutants, the insertion of IS2/600 within the distal binding site of the negative regulator NagC leads to ChbR-dependent cellobiose-inducible expression of the operon. These studies underscore the significance of strain background, specifically the diversity of transposable elements, in the evolution of novel metabolic functions. Constitutive expression of the chb operon also enables utilization of the aromatic ß-glucosides arbutin and salicin, implying that the chb structural genes are inherently promiscuous.

Towards a peptide-based vaccine against Shigella sonnei: A subtractive reverse vaccinology based approach.

Shigella sonnei is one of the major causes of shigellosis in technically advanced countries and reports of its unprecedented increase are published from the Middle East, Latin America, and Asia. The pathogen exhibits resistance against first and second line antibiotics which highlights the need for the development of an effective broad-spectrum vaccine. A computational based approach comprising subtractive reverse vaccinology was used for the identification of potential peptide-based vaccine candidates in the proteome of S. sonnei reference strain (53G). The protocol revealed three essential, host non-homologous, highly virulent, antigenic, conserved and adhesive vaccine proteins: TolC, PhoE, and outer membrane porin protein. The cellular interactome of these proteins supports their direct and indirect involvement in biologically significant pathways, essential for pathogen survival. Epitope mapping of these candidates reveals the presence of surface exposed 9-mer B-cell-derived T-cell epitopes of an antigenic, virulent, non-allergen nature and have broad-spectrum potency. In addition, molecular docking studies demonstrated the deep binding of the epitopes in the binding groove and the stability of the complex with the most common binding allele in the human population, DRB1*0101. Future characterization of the screened epitopes in order to further investigate the immune protection efficacy in animal models is highly desirable.

Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent Biosensor Platform.

In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Shigella sonnei. Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t) values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively) had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity (KD) of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor platform consisted of three elements: (1) 5'amine-SS-4 modification in a 96-well type microtiter plate surface (N-oxysuccinimide, NOS) as capture probes; (2) the incubation with S. sonnei and test microbes in functionalized 96 assay wells in parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate S. sonnei from other enteric species such as E. coli, Salmonella typhimurium and other Shigella species (S. flexneri, S. boydii). In this study, we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.

[SONNEI DYSENTERY MORBIDITY IN KHABAROVSK AND KHABAROVSK REGION DUE TO ATYPICAL MANNITOL-NEGATIVE CAUSATIVE AGENT].

AIM: Determine features of epidemic process (EP) of Sonnei dysentery in Khabarovsk Region in 2012 - 2014 due to atypical causative agent. MATERIALS AND METHODS: Detailed characteristics of 161 cultures of Shigella sonnei isolated from 81 patients from epidemic focus in children boarding school in Bikin as well as from 22 patients from sporadic and group foci of dysenteryin Khabarovsk (biochemical type, colicin-genotype, spectrum of drug resistance) is given. Molecular-biologic subtyping was carried out for 11 strains by Pulsed Field Gel Electrophoresis method (PFGE). RESULTS: Materials of observation of a prolonged foci of Sonnei dysentery with contact-domestic transmission route of the infection in children boarding house for disabled (October 2012 - September 2014) are presented. The diseases are etiologically connected with atypical mannitol- negative types of shigella isolated for the first time in 40 years of observation in Khabarovsk region. Epidemic process of shigellosis was supported by prolonged carriership of the causative agent in patients and special contingent ofthe nursing home. Shigella cultures isolated in the focus belonged to the same colicin-genotype and 2 distinct drug resistance clones, but a single genotype established by PFGE method. CONCLUSION: Results of the studies give evidence on the importance of determi- nation of traditional phenotypic and contemporary genotypic variants of shigella and the neces- sity of search for arguments, additional methodic approaches for establishing similarities and differences of shigella isolates from within the same outbreak of the diseases as well as for com- parison of strains circulating in different territories.

Expression of different bacterial cytotoxins is controlled by two global transcription factors, CRP and Fis, that co-operate in a shared-recruitment mechanism.

Pet is a cytotoxic autotransporter protein secreted by the pathogenic enteroaggregative Escherichia coli strain 042. Expression of Pet is co-dependent on two global transcription regulators: CRP (cyclic AMP receptor protein) and Fis (factor for inversion stimulation). At the pet promoter CRP binds to a single site centred at position -40.5 upstream of the start site for transcription. Due to the suboptimal positioning of this site, CRP alone activates transcription poorly and requires Fis to bind upstream to promote full activation. Here, we show that CRP and Fis control the expression of other important autotransporter toxins, namely Sat from uropathogenic E. coli (UPEC) and SigA from Shigella sonnei, and that this regulation has been conserved in different pathogens. Furthermore, we investigate the mechanism of Fis-mediated co-activation, exploiting a series of semi-synthetic promoters, with similar architecture to the pet promoter. We show that, when bound at position -40.5, CRP recruits RNA polymerase inefficiently and that Fis compensates by aiding polymerase recruitment through a direct protein-protein interaction. We demonstrate that other suitably positioned upstream transcription factors, which directly recruit RNA polymerase, can also compensate for the inappropriate positioning of CRP. We propose that this is a simple 'shared-recruitment' mechanism, by which co-dependence of promoters on two transcription factors could evolve.

First evidence for a covalent linkage between enterobacterial common antigen and lipopolysaccharide in Shigella sonnei phase II ECALPS.

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

Activity spectrum of colicins produced by Shigella sonnei and genetic mechanism of colicin resistance in conspecific S. sonnei strains and Escherichia coli.

Colicin-mediated killing is an example of allelopathy, which has been found among several bacteria. Screening of 42 strains of Shigella sonnei isolated from diarrheal patients revealed that 39 (93%) S. sonnei strains were positive for colicin production against Escherichia coli DH5α. In the PCR-based detection of the colicin types, 36 (92.3%) were identified as E3, 2 (5.1%) as E3 and E8, and 1 (2.6%) as E3 and E2. Representative S. sonnei strains producing heterologous colicins exhibited antagonism against diarrheagenic Escherichia coli (DEC) groups. Although it is known that mutation in the colicin receptor renders the host resistant to colicin, there is a dearth of information on the genetic characterization of such mutants. In the fluctuation test, colicin-resistant E. coli mutants were found to occur spontaneously at the rates of 2.51 × 10(-8) and 5.52 × 10(-8) per generation when exposed to colicins E3 and E8 and colicins E3 and E2, respectively. Genotypic characterization of colicin-resistant E. coli (EC(Cr)) and S. sonnei (SS(Cr)) strains displayed mutations in the btuB gene, which encodes the receptor for vitamin B12 uptake. This gene was interrupted by various insertion sequences, such as IS1, IS2, and IS911. Complementation of EC(Cr) and SS(Cr) with plasmid-borne btuB (pbtuB) accomplished restoration of the colicin-susceptible phenotype. The vitamin B12 uptake assay gave an insight into the physiological relevance of the btuB mutation. Our studies provide insights into the latent influence of S. sonnei colicins in governing the existence of some of the shigellae and all of the DEC and the genetic mechanism underlying the emergence of resistance.

Structural mechanism of bacteriophage lambda tail's interaction with the bacterial receptor.

Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system's biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.

RodZ regulates the post-transcriptional processing of the Shigella sonnei type III secretion system.

The expression of the type III secretion system-a main determinant of virulence in Shigella-is controlled by regulator cascades VirF-InvE (VirB) and CpxAR two-component system. A screen for mutants that restore virulence in the cpxA background led to the isolation of a mutant of rodZ, a cytoskeletal protein that maintains the rod-shaped morphology of bacilli. InvE is normally repressed at 30 °C because of decreased messenger RNA (mRNA) stability, but rodZ mutants markedly increase invE-mRNA stability. Importantly, the inhibition of InvE production by RodZ can be genetically separated from its role in cell-shape maintenance, indicating that these functions are distinguishable. Thus, we propose that RodZ is a new membrane-bound RNA-binding protein that provides a scaffold for post-transcriptional regulation.

From an in vivo to an in vitro relative potency (IVRP) assay to fully characterize a multicomponent O-antigen based vaccine against Shigella.

Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.

Exploring the mechanisms by which camel lactoferrin can kill Salmonella enterica serovar typhimurium and Shigella sonnei.

There is a continuously increasing pressure associated with the appearance of Salmonella enterica Serovar typhimurium (S. typhimurium) and Shigella sonnei (S. sonnei) that have developed pathogenic multiple antibiotic resistance and the cost of cure and control of these enterobacteriaceae infections increases annually. The current report for first time demonstrated the distinguished antimicrobial action of camel lactoferrin (cLf) obtained from the milk of different clans of camel in Saudi Arabia against S. typhimurium and S. sonnei. These cLf subtypes showed comparable antimicrobial potential when tested against the two bacterial strains but were superior to either bovine (bLf) or human lactoferrin (hLf). The synergism between lactoferrins and antibiotics concerning their antibacterial efficacies against the two bacterial strains was evident. Exploring mechanisms by which camel lactoferrin can kill S. typhimurium and S. sonnei revealed that cLf affects bacterial protein profile. Besides, it interacts with bacterial lipopolysaccharides (LPS) and numerous membrane proteins of S. typhimurium and S. sonnei, with each bacterial strain possessing distinctive binding membrane proteins for lactoferrin. Furthermore, as evidenced by electron microscopy analysis, cLf induces extracellular and intracellular morphological changes in the test bacterial strains when used alone or in combination treatment with antibiotics. Lactoferrin and antibiotics combination strongly disrupts the integrity of the bacterial cells and their membranes. Therefore, cLf can kill S. typhimurium and S. sonnei by four different mechanisms, such as iron chelation, affecting some bacterial proteins, binding to bacterial LPS and membrane proteins, and impairing the integrity of the bacterial cells and their membranes.

Involvement of RNA chaperone hfq in the regulation of antibiotic resistance and virulence in Shigella sonnei.

The host factor for RNA phage Qß replicase (Hfq) is a crucial post-transcriptional regulator in many bacterial pathogens, facilitating the interaction between small non-coding RNAs (sRNAs) and their target mRNAs. Studies have suggested that Hfq plays a role in antibiotic resistance and virulence in bacteria, although its functions in Shigella are not fully understood. In this study, we investigated the functional roles of Hfq in Shigella sonnei (S. sonnei) by constructing an hfq deletion mutant. Our phenotypic assays showed that the hfq deletion mutant was more sensitivity to antibiotics and had impaired virulence. Transcriptome analyses supported the results concerning the phenotype of the hfq mutant and showed that differentially expressed genes were mainly enriched in the KEGG pathways two-component system, ABC transporters, ribosome, and Escherichia coli biofilm formation. Additionally, we predicted eleven novel Hfq-dependent sRNAs, which were potentially involved in the regulation of antibiotic resistance and/or virulence in S. sonnei. Our findings suggest that Hfq plays a post-transcriptional role in regulating antibiotic resistance and virulence in S. sonnei, and could provide a basis for future studies on Hfq-sRNA-mRNA regulatory networks in this important pathogen.

The detrimental influence of bacteria (E. coli, Shigella and Salmonella) on the degradation of organic compounds (and vice versa) in TiO2 photocatalysis and near-neutral photo-Fenton processes under simulated solar light.

TiO2 photocatalytic and near-neutral photo-Fenton processes were tested under simulated solar light to degrade two models of natural organic matter - resorcinol (R) (which should interact strongly with TiO2 surfaces) and hydroquinone (H) - separately or in the presence of bacteria. Under similar oxidative conditions, inactivation of Escherichia coli, Shigella sonnei and Salmonella typhimurium was carried out in the absence and in the presence of 10 mg L(-1) of R and H. The 100% abatement of R and H by using a TiO2 photocatalytic process in the absence of bacteria was observed in 90 min for R and in 120 min for H, while in the presence of microorganisms abatement was only of 55% and 35% for R and H, respectively. Photo-Fenton reagent at pH 5.0 completely removed R and H in 40 min, whereas in the presence of microorganisms their degradation was of 60% to 80%. On the other hand, 2 h of TiO2 photocatalytic process inactivated S. typhimurium and E. coli cells in three and six orders of magnitude, respectively, while S. sonnei was completely inactivated in 10 min. In the presence of R or H, the bacterial inactivation via TiO2 photocatalysis was significantly decreased. With photo-Fenton reagent at pH 5 all the microorganisms tested were completely inactivated in 40 min of simulated solar light irradiation in the absence of organics. When R and H were present, bacterial photo-Fenton inactivation was less affected. The obtained results suggest that in both TiO2 and iron photo-assisted processes, there is competition between organic substances and bacteria simultaneously present for generated reactive oxygen species (ROS). This competition is most important in heterogeneous systems, mainly when there are strong organic-TiO2 surface interactions, as in the resorcinol case, suggesting that bacteria-TiO2 interactions could play a key role in photocatalytic cell inactivation processes.

Pretreatment of epithelial cells with rifaximin alters bacterial attachment and internalization profiles.

Rifaximin is a poorly absorbed semisynthetic antibiotic derivative of rifampin licensed for use in the treatment of traveler's diarrhea. Rifaximin reduces the symptoms of enteric infection, often without pathogen eradication and with limited effects on intestinal flora. Epithelial cells (HEp-2 [laryngeal], HCT-8 [ileocecal], A549 [lung], and HeLa [cervical]) were pretreated with rifaximin (or control antibiotics) prior to the addition of enteroaggregative Escherichia coli (EAEC). EAEC adherence was significantly reduced following rifaximin pretreatment compared to pretreatment with rifampin or doxycycline for three of the four cell lines tested. The rifaximin-mediated changes to epithelial cells were explored further by testing the attachment and internalization of either Bacillus anthracis or Shigella sonnei into A549 or HeLa cells, respectively. The attachment and internalization of B. anthracis were significantly reduced following rifaximin pretreatment. In contrast, neither the attachment nor the internalization of S. sonnei was affected by rifaximin pretreatment of HeLa cells, suggesting that rifaximin-mediated modulation of host cell physiology affected bacteria utilizing distinct attachment/internalization mechanisms differently. In addition, rifaximin pretreatment of HEp-2 cells led to reduced concentrations of inflammatory cytokines from uninfected cells. The study provides evidence that rifaximin-mediated changes in epithelial cell physiology are associated with changes in bacterial attachment/internalization and reduced inflammatory cytokine release.

Diverse pathways for salicin utilization in Shigella sonnei and Escherichia coli carrying an impaired bgl operon.

Utilization of the aryl-ß-glucosides salicin or arbutin in most wild-type strains of E. coli is achieved by a single-step mutational activation of the bgl operon. Shigella sonnei, a branch of the diverse E. coli strain tree, requires two sequential mutational steps for achieving salicin utilization as the bglB gene, encoding the phospho-ß-glucosidase B, harbors an inactivating insertion. We show that in a natural isolate of S. sonnei, transcriptional activation of the gene SSO1595, encoding a phospho-ß-glucosidase, enables salicin utilization with the permease function being provided by the activated bgl operon. SSO1595 is absent in most commensal strains of E. coli, but is present in extra-intestinal pathogens as bgcA, a component of the bgc operon that enables ß-glucoside utilization at low temperature. Salicin utilization in an E. coli bglB laboratory strain also requires a two-step activation process leading to expression of BglF, the PTS-associated permease encoded by the bgl operon and AscB, the phospho-ß-glucosidase B encoded by the silent asc operon. BglF function is needed since AscF is unable to transport ß-glucosides as it lacks the IIA domain involved in phopho-relay. Activation of the asc operon in the Sal(+) mutant is by a promoter-up mutation and the activated operon is subject to induction. The pathway to achieve salicin utilization is therefore diverse in these two evolutionarily related organisms; however, both show cooperation between two silent genetic systems to achieve a new metabolic capability under selection.

Long-Term Monitoring of Noxious Bacteria for Construction of Assurance Management System of Water Resources in Natural Status of the Republic of Korea.

Climate change is expected to affect not only availability and quality of water, the valuable resource of human life on Earth, but also ultimately public health issue. A six-year monitoring (total 20 times) of Escherichia coli O157, Salmonella enterica, Legionella pneumophila, Shigella sonnei, Campylobacter jejuni, and Vibrio cholerae was conducted at five raw water sampling sites including two lakes, Hyundo region (Geum River) and two locations near Water Intake Plants of Han River (Guui region) and Nakdong River (Moolgeum region). A total 100 samples of 40 L water were tested. Most of the targeted bacteria were found in 77% of the samples and at least one of the target bacteria was detected (65%). Among all the detected bacteria, E. coli O157 were the most prevalent with a detection frequency of 22%, while S. sonnei was the least prevalent with a detection frequency of 2%. Nearly all the bacteria (except for S. sonnei) were present in samples from Lake Soyang, Lake Juam, and the Moolgeum region in Nakdong River, while C. jejuni was detected in those from the Guui region in Han River. During the six-year sampling period, individual targeted noxious bacteria in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five Korea's representative water environments make it necessary to establish the chemical and biological analysis for noxious bacteria and sophisticated management systems in response to climate change.