Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

Role of lipopolysaccharide in the receptor function for bacteriophage Ox2

Abstract The sensitivity of Salmonella typhimurium as well as of Escherichia coli to the bacteriophage Ox2 was found to require, in addition to the OmpA protein, a certain type of rough LPS. Heptoseless mutants were resistant and unable to adsorb the phage. Mutants with less defective LPS chemotype were sensitive and could, except the second most defective chemotypes, adsorb Ox2. However, isolated LPS-free OmpA protein could bind the phage, and this binding could not be increased by adding LPS.     

Complete genome sequence of Salmonella enterica serovar typhimurium bacteriophage SPN1S

To understand the interaction between the host of pathogenic Salmonella enterica serovar Typhimurium and its bacteriophage, we isolated the bacteriophage SPN1S. It is a lysogenic phage in the Podoviridae family and uses the O-antigen of lipopolysaccharides (LPS) as a host receptor. Comparative genomic analysis of phage SPN1S and the S. enterica serovar Anatum-specific phage ε15 revealed different host specificities, probably due to the low homology of host specificity-related genes. Here we report the complete circular genome sequence of S. Typhimurium-specific bacteriophage SPN1S and show the results of our analysis.     

Isolation and characterization of a bacteriophage specific for the lipopolysaccharide of rough derivatives of Pseudomonas aeruginosa strain PAO

A lipopolysaccharide (LPS)-defective (rough) mutant of Pseudomonas aeruginosa PAO was isolated by selection for resistance to the LPS-specific phage E79. The LPS of this mutant, AK-1012, lacked the O-antigenic side chain-specific amino sugar fucosamine as well as the core-specific sugars glucose and rhamnose. Using this strain, we isolated and characterized a phage, phi PLS27, which is specifically inactivated upon incubation with LPS extracted from rough mutants of P. aeruginosa PAO. phi PLS27 was found to be a Bradley type C phage and was very similar to coliphage T7 in a number of properties, including size, buoyant density, mass, and the number of structural proteins.     

Influence of O Side Chains on the Attachment of the Felix O-1 Bacteriophage to Salmonella Bacteria

The adsorption rate constant (ARC) of the Felix O-1 (FO) bacteriophage to sensitive Salmonella strains was used to determine the effect of variations in surface antigens on phage attachment. The N-acetylglucosamine of the common-core polysaccharide of the Salmonella lipopolysaccharide (LPS) was found to be an essential part of the receptor for the FO phage in conformation with earlier reports. It was found that (i) the ARC was low for strains having O side chains containing two or three non core monosaccharides, (ii) the ARC varied when the O side chain contained no, or only one, noncore monosaccharide, (iii) the ARC was high when the O side chain contained only one repeating unit, and (iv) the ARC was high to mutants of chemotype Ra in which the N-acetylglucosamine was the terminal sugar of the LPS. Since a good correlation was found between the ARC of the FO phage and the phage-inactivating capacity of phenol water-extracted LPS, the results suggest that only the structure and composition of the LPS determines the adsorption rate of the FO phage. The phage-inactivating capacity of LPS from the Ra mutants increased in parallel with higher glucosamine contents in the core polysaccharide. In smooth strains having long and numerous O side chains, the access of the FO phage to its receptor is probably blocked by the presence of the side chains, whereas short and numerous side chains or T1 side chains do not interfere with the FO attachment.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

The Molecular and Genetic Basis of Repeatable Coevolution between Escherichia coli and Bacteriophage T3 in a Laboratory Microcosm

The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.     

Plasmid transduction by Bacillus anthracis bacteriophage CP54

Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.     

The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli

AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC) and enteropathogenic E. coli (EPEC). METHODS AND RESULTS: A total of 100 strains of E. coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR. Fifty-four per cent produced an amplicon with the R3 primer set. Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25. However, 46% of enteroaggregative E. coli and 50% of enteropathogenic E. coli were also shown to have an R3 LPS-core structure. CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Complete genome sequence of Salmonella enterica serovar Typhimurium bacteriophage SPN3UB

Salmonella is one of the major pathogenic bacteria that cause food poisoning. To elucidate the host infection mechanism of Salmonella enterica serovar Typhimurium-targeting phages, the bacteriophage SPN3UB was isolated from a chicken fecal sample. This phage belongs morphologically to the Siphoviridae family and infects the host via the O antigen of lipopolysaccharide (LPS). To further understand its infection mechanism, we completely sequenced and analyzed the genome. Here, we announce its complete genome sequence and report major findings from the genomic analysis results.     

Characterization of a bacteriophage related to R-type pyocins.

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.     

1株产L-天冬氨酸酶大肠埃希菌噬菌体分离和生理特性初步研究

对产L-天冬氨酸酶大肠埃希菌噬菌体进行分离和生理特性研究,有助于为生产过程中噬菌体污染的防治提供指导。采用双层平板法对噬菌体进行分离纯化。利用透射电镜观察噬菌体形态。进行噬菌体全基因组测序和比对。通过测定不同处理条件下噬菌体活性,研究温度、pH、有机溶剂氯仿、去垢剂SDS对噬菌体的影响。从噬菌体污染的L 天冬氨酸酶生产菌种大肠埃希菌HY-05C发酵培养液中分离出1株噬菌体。电镜结果表明,该噬菌体由呈多面体对称的头部和极短的尾部构成。基因组测序和比对结果表明,噬菌体与T7样噬菌体的相似性最高。生理特性研究表明,噬菌体对高温和去垢剂SDS敏感,对有机溶剂氯仿不敏感;最适pH为7.0,碱性条件下活力较为稳定,酸性条件容易失活。噬菌体保藏编号为CICC 80001。     

Ampicillin-resistant mutants of Escherichia coli K-12 with lipopolysaccharide alterations affecting mating ability and susceptibility to sex-specific bacteriophages

Mutations from moderate (class I) to high (class III) ampicillin resistance in a male and a female strain of Escherichia coli K-12 have been found to be accompanied by surface alterations, first demonstrated as hindrance in the formation of mating pairs. These changes have now been studied with the ribonucleic acid phage MS2, and especially with the "female-specific" phage phiW. Several class III mutations in male and female strains were found to make the cells susceptible to phage phiW and to reduce their abilities to form mating pairs. Spontaneous phage phiW-resistant mutants isolated from class III strains were found also to have acquired changes in ampicillin resistance and ability to form mating pairs. One mutant had reverted to parental class I type in all three properties. Lipopolysaccharides (LPS) prepared from phiW-sensitive class III strains inactivated the phage in vitro, whereas LPS from phage-resistant strains had no effect. Carbohydrate analyses of LPS preparations showed that two class III mutants, compared to their parental strains, had lost significant parts of the rhamnose, galactose, and glucose from the LPS. One of the phage phiW-resistant mutants showed a partial restoration of its carbohydrate composition. Other phiW-resistant mutants showed, instead, further losses of carbohydrates in their LPS. It is suggested that genes exist which simultaneously mediate a female-specific mating site, ampicillin resistance, and the receptors for phage phiW.     

Structure of the lipid-containing bacteriophage PRD1: disruption of wild-type and nonsense mutant phage particles with guanidine hydrochloride.

The lipid-containing bacteriophage PRD1 was disrupted, and the subviral particles were studied. Guanidine treatment released two phage proteins (P3 and P5). These proteins form the polyhedral capsid. The remaining phage proteins were associated with the phage membrane vesicle. The vesicle was capable of forming a tubular structure. The isolated phage membrane vesicles aggregated readily. We found that aggregation and tube formation were associated with specific phage proteins (P11 and P18, respectively) by using protease treatment and an analysis of nonsense mutant phage particles. In addition, the possibility that free vesicles might be precursors to empty virions was studied.     

鲍曼不动杆菌裂解性噬菌体LZ35的 分离及全基因组分析

摘要：目的 通过分离鉴定鲍曼不动杆菌噬菌体并进行遗传信息分析,为今后噬菌体用于治疗鲍曼不动杆菌引起的感染提供依据。方法 以鲍曼不动杆菌临床分离株为宿主菌,从医院污水中分离鲍曼不动杆菌噬菌体并进行纯化、电镜观察形态特征、提取噬菌体DNA,进行全基因组测序,分析全基因组的结构特征,比较基因组分析其进化关系。结果 分离到鲍曼不动杆菌裂解性噬菌体LZ35,电镜观察显示,该噬菌体属于有尾噬菌体目肌尾病毒科。基因组全长44 885 bp,G＋C含量为37.95%,含有83个开放阅读框,其中22个编码序列可预测其功能,61个编码序列为未知基因。噬菌体LZ35的基因组与鲍曼不动杆菌噬菌体IME-AB2和YMC-13-01-C62具有很高的同源性（分别为97%和99%）,与鲍曼不动杆菌噬菌体YMC11/12/R1215的进化关系最近。结论 以鲍曼不动杆菌临床分离株为宿主菌,分离到鲍曼不动杆菌裂解性噬菌体LZ35,明确了其形态和基因组特征,为防治噬菌体疗法奠定基础。     

Attachment of a long-tailed Rhizobium bacteriophage to the pili of its host

Bacterial strain 16-12 was isolated from the root nodules of lupines and was found to be mitomycin C-inducible for the production of a bacteriophage (“16-12-1”) with a long noncontractile tail. The phage was found to attach with a fork-like terminal tail structure to the pili of strain 16-12. In addition, it was also found adsorbed to the bacterial cell poles. It is suggested that phage 16-12-1 may be pilus dependent.     

The serological response to Verocytotoxigenic Escherichia coli in patients with haemolytic uraemic syndrome

AIMS: To screen sera from 80 patients with clinical haemolytic uraemic syndrome (HUS) and serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157, for antibodies to Verocytotoxin-producing Escherichia coli (VTEC) belonging to serogroups O5, O26, O104, O111, O128, O145, O153 and O165. METHODS AND RESULTS: Sera were screened by an LPS-based ELISA and SDS-PAGE/immunoblotting. None of the 80 sera contained antibodies binding to long-chain LPS of any of the LPS types employed; however, nine sera contained antibodies binding to R3 LPS-core epitopes. CONCLUSIONS: The presence of patients' serum antibodies to the LPS of E. coli O157, in the absence of antibodies to the LPS of a range of other VTEC, demonstrated that cases of HUS may be caused by strains of O157 VTEC alone and that concurrent infection with multiple strains of VTEC is not a prerequisite for cases of HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibodies to long-chain LPS of VTEC other than O157 were not detected, and so there was no evidence of infection with VTEC belonging to more than one serogroup. The results of immunoassays such as ELISAs and micro-agglutinations must take into consideration antibodies binding to R3 epitopes located on LPS-core.