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Several biocides commonly used in disinfection processes as antibacterial and antifungal agents were tested for activity against MS2 and K coliphages. MS2 was resistant to most biocides; only glutaraldehyde (0.5%) and peracetic acid (1%) achieved a 4-log(10) titer reduction in 20 min. In contrast, K phage was sensitive to most biocides, being resistant only to phenol (2%) and chlorhexidine (1%).
We have shown previously that Klebsiella pneumoniae receptors for coliphages T3 and T7 also mediate mannose-inhibitable adherence to human epithelial cells and protect bacteria from phagocytosis and intracellular killing by human polymorphonuclear cells. In this paper we analyze the possible role of such mannose-inhibitable adhesins and T3-T7 receptors (MIAT) in K. pneumoniae intraperitoneal pathogenicity for mice. We showed that intraperitoneal pathogenicity for mice of four different Klebsiella strains (one laboratory and three wild-type) that carry the MIAT was approximately 60-fold higher than that of four derivative strains that lost such receptors by spontaneous mutation. The MIAT could be repressed by Klebsiella phage AP3 lysogenic conversion. Two laboratory and two wild-type strains converted by phage AP3 were also approximately 60-fold less pathogenic for mice than parental strains and showed a pathogenicity level equal to that of the MIAT-negative mutants. Studies of protection in mice with anti-whole cell antisera showed that passive immunization against MIAT-positive cells was more protective than immunization against MIAT-negative cells. Studies of protection in mice by both active and passive immunization with lipopolysaccharide and purified outer membrane proteins have shown that the proteins are the most protective outer membrane components. Since it has been shown previously that the Klebsiella receptors for T3-T7 have a proteic component and that an outer membrane protein is missing in the strains resistant to T3-T7 (C. Pruzzo et al., in R. C. Berkely (ed.), Microbial Adhesion to Surfaces, 1980); the latter finding further supports the role of MIAT in the pathogenicity of Klebsiella for mice.
A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.
We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon. The vector carries the strong leftward promoter (pL) of coliphage lambda as well as the cI857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.
We have developed a new research tool for the study of S-adenosylmethionine (AdoMet) metabolism by cloning the coliphage T3 AdoMet hydrolase (AdoMetase; EC 3.3.1.2) gene into the M13mp8 expression vector. The recombinant bacteriophage clones expressed an AdoMetase activity in Escherichia coli like that found in T3-infected cells. High levels of AdoMetase expression impaired AdoMet-mediated activities such as dam and dcm methylase-directed DNA modifications and the synthesis of spermidine from putrescine. Expression vectors containing the cloned AdoMetase gene thus provide an alternate approach to the use of chemical inhibitors or mutants defective in AdoMet biosynthesis to probe the effect of AdoMet limitation.
Assembly of tail fibers of coliphage T4 requires the action of helper proteins. In the absence of one of these, protein 38 (p38), p37, constituting the distal part of the long tail fiber, fails to oligomerize. In the absence of the other, p57, p34 (another component of the long tail fiber), p37, and p12 (the subunit of the short tail fiber) remain unassembled. p38 can be replaced by the Tfa (tail fiber assembly) protein (pTfa) of phage lambda, which has the advantage of remaining soluble even when produced in massive amounts. The mechanisms of action of the helpers are unknown. As a first step towards elucidation of these mechanisms, p57 and pTfa have been purified to homogeneity and have been crystallized. The identity of gene 57 (g57), not known with certainty previously, has been established. The 79-residue protein p57 represents a very exotic polypeptide. It is oligomeric and acidic (an excess of nine negative charges). It does not contain Phe, Trp, Tyr, His, Pro, and Cys. Only 25 N-terminal residues were still able to complement a g57 amber mutant, although with a reduced efficiency. In cells overproducing the protein, it assumed a quasi-crystalline structure in the form of highly ordered fibers. They traversed the cells longitudinally (and thus blocked cell division) with a diameter approaching that of the cell and with a hexagonal appearance. The 194-residue pTfa is also acidic (an excess of 13 negative charges) and is likely to be dimeric.
Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.
CTXphi is a filamentous bacteriophage that encodes cholera toxin. CTXphi infection of its host bacterium, Vibrio cholerae, requires the toxin-coregulated pilus (TCP) and the products of the V. cholerae tolQRA genes. Here, we have explored the role of OrfU, a predicted CTXphi minor coat protein, in CTXphi infection. Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXphi genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXphi infection as well as playing a role in CTXphi assembly and release. Deletion of orfU from CTXphi dramatically reduced the number of CTXphi virions detected in supernatants from CTXphi-bearing cells. This defect was complemented by expression of orfU in trans, thereby confirming a role for this gene in CTXphi assembly and/or release. To evaluate the requirement for OrfU in CTXphi infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect V. cholerae, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V. cholerae in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXphi pIII, we propose to rename OrfU as pIII(CTX). Our data also provide new evidence for a conserved pathway for filamentous phage infection.
The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.
A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.
Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C. Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.
Genetic and DNA heteroduplex analyses of lambda imm22 hybrid phages were used to compare the Salmonella bacteriophage P22 and coliphage lambda genes which control late gene regulation and lysis. Homologous DNA sequences were correlated with P22 gene 23 and lambda gene Q (late gene regulation) and with P22 gene 13 and lambda gene S (lysis control). Nonhomologous DNA sequences were correlated with P22 gene 19 and lambda gene R (lysozyme and endolysin) and with the region encoding the P22 alpha and lambda 6S transcripts.
We conducted a series of experiments to compare the survival of Escherichia coli, feline calicivirus, and F-specific coliphage MS2 on lettuce and cabbage with and without disinfection. Inoculated produce was held at 4, 25, or 37 degrees C for 21 days or was treated with different concentrations of sodium bicarbonate, chlorine bleach, peroxyacetic acid, or hydrogen peroxide. Survival was measured by the decimal reduction value (time to 90% reduction in titer) and the change in log titers of the test organisms. A stronger correlation of survival measures was observed between feline calicivirus and MS2 than between E. coli and either of the viral agents at 25 and 37 degrees C. The maximum time to detection limit for MS2 at all temperatures was 9 days, whereas feline calicivirus was detected for a maximum of 14 days at 4 degrees C. In contrast, E. coli was detectable for 21 days at 4 and 25 degrees C and for 14 days at 37 degrees C. Significant increases in E. coli titer occurred within the first 5 days, but virus titers decreased steadily throughout the experiments. E. coli was also highly susceptible to all disinfectants except 1% sodium bicarbonate and 50 ppm chlorine bleach, whereas the viruses were resistant to all four disinfectants.
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