Deletion Map of the Escherichia coli K-12 Sex Factor F: the Order of Eleven Transfer Cistrons

A series of Hfr deletion mutants was isolated. These mutants contain deletions which extend from a lambda prophage into an Flac which is integrated into the gal operon. Transfer-deficient deletion mutants were found to fall into four different phenotypic groups when tested for male- and female-specific phage resistance. Conjugational and transductional complementation tests with Flac point mutants deficient in transfer (tra(-)) were performed, and the order of 11 tra cistrons was determined. The tra genes are all located between an F gene for the inhibition of female-specific phages and the transposed lac operon originally carried by the Flac. The order of genes in the Hfr studied was established to be: proC... phi(II) (R)... traJ traA traE traK traB traC traF traH traG traD traI...lac...attlambda...bio.     

Studies on Sex Pili: Mutants of the Sex Factor F in Escherichia coli Defective in Bacteriophage-Adsorbing Function of F Pili

Ultraviolet irradiation or nitrosoguanidine treatment of Escherichia coli K-12 strain JE3100 (F'(8)/fla pil) led to the isolation of six mutants defective in F pili function. The defects were shown to be caused by mutations in the F factor. The mutants retained conjugal fertility, although they were less efficient than parental F'(8) strain, and continued to synthesize F pili. Three of the mutants (strains KE196, 198, and 200) had lost sensitivity to male-specific MS2 phage, and the other three (strains KE161, 163, and 164) were insensitive to Qbeta and f1 as well as MS2 phages. F pili on strains KE196, 198, and 200 cells continued to adsorb MS2 phage, whereas those of strains KE161, 163, and 164 did not adsorb MS2 phage. The correlation of the mutant phenotypes with those of other F mutants reported in the literature is discussed.     

Derepression of F factor function in Salmonella typhimurium

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.     

Variant pili produced by mutants of the Flac plasmid

Transfer-proficient Flac mutants with reduced abilities to plate various F-specific phages were isolated, either by selection after mutagenesis, or as revertants of Flac traA mutants. In many of the mutants pilus-related properties were altered, including physical adsorption of R17 phage, the number of pili per cell and the outgrowth/retraction equilibrium. Complementation studies showed that the mutations were in traA, suggesting that specific alterations in the amino-acid sequence of the pilin subunit protein were responsible for the altered pilus properties. Complementation between the Flac traA mutants and the derepressed plasmid R100-1 restored phage sensitivity in some cases, suggesting that the incorporation of both mutant and R100-1 subunits into the pilus structure may result in conformational changes which increase the capacity of the pilus to interact with phages.     

Escherichia coli females defective in conjugation and in adsorption of a single-stranded deoxyribonucleic acid phage

We predicted that, among mutants resistant to infection by single-stranded deoxyribonucleic acid viruses, there would be some also resistant to "infection" by single-stranded conjugal deoxyribonucleic acid. Approximately 5% of the Escherichia coli K-12 females selected for resistance to phage ST-1 were defective as recipients in conjugation. These spontaneous mutants fell into two classes. Type A accepted both plasmid and chromosomal markers at greatly reduced frequencies (<10(-6) of normal for at least one strain), formed "rough" colonies, and (unlike their parent) were nonflagellated. Type B strains accepted both chromosomal and plasmid markers at reduced frequencies (10(-2) to 10(-1) of normal), were temperature sensitive for growth, and showed increased susceptibility towards antibiotics and deoxycholate. Both classes of mutants also were resistant to certain female-specific viruses.     

Inhibition of Formation of Escherichia coli Mating Pairs by f1 and MS2 Bacteriophages as Determined with a Coulter Counter

The effect of male-specific filamentous deoxyribonucleic acid (f1) and isometric ribonucleic acid (MS2) bacteriophages on the formation of mating pairs in Escherichia coli conjugation was examined directly in the Coulter counter. When a sufficient multiplicity of infection (MOI) was used, the f1 phage immediately and completely inhibited the formation of mating pairs. On the other hand, the MS2 phage at a relatively high MOI also inhibited the formation of mating pairs significantly although not completey. The inhibitory effect of MS2 phage was dependent on the time of addition and the MOI used. At relatively low MOI (<20), the MS2 phage showed some inhibitory effect when added to a male culture prior to mixing with females, whereas no effect was observed when phages were added after mating pair formation had already commenced. At a high MOI (>400) MS2 phage disrupted the mating pairs already formed. Some preformed mating pairs were resistant to the high MOI of MS2 phages, however, and the "sensitive" (to high MOI) mating pairs seem to mature into "resistant" mating pairs as a function of time. We conclude that the tip of an F pilus is the specific attachment site for mating. The following process of mating pair formation has been formulated by deduction. (i) The sides of F pili weakly contact female cells, (ii) then the tips of F pili attach to the specific receptor sites to form initial mating pairs, and (iii) those pairs mature into mating pairs that are resistant to the high MOI of MS2 phages. The high MOI of MS2 prevents the first step, whereas f1 phages affect the second step-the binding between the tips of F pili and the receptor sites.     

Streptomycin-resistant Escherichia coli mutant temperature sensitive for the production of Qbeta-infective particles.

A streptomycin-resistant Escherichia coli mutant has been isolated that is temperature sensitive for Qbeta phage, but not for the group I RNA phages f2, MS2, and R17. The growth of Qbeta in the mutant at the nonpermissive temperature (42 degrees C) results in the release of a near-normal burst of noninfectious particles that cosediment with Qbeta in a sucrose gradient. It is assumed that the mutant is defective at elevated temperatures in the suppression of nonsense codons, thereby producing Qbeta-like particles which are noninfectious because of the lack of the read-through protein A1.     

Synthesis of Indicator Strains and Density of Ribonucleic Acid-Containing Coliphages in Sewage

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

A method for the enumeration of male-specific bacteriophages in sewage

H avelaar , A.H. & H ogeboom , W.M. 1984. A method for the enumeration of male-specific bacteriophages in sewage. Journal of Applied Bacteriology 56 , 439–447.
Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F^--salmonellas—usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimu-rium phage type 3, plaque counts in secondary effluent were found to be in the range of 60–8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiona carrying F'42 lac . A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F^- E. coli but not F^- Salmonella were isolated using this host strain.     

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

A physiological role for tRNA nucleotidyltransferase during bacteriophage infection

Bacteriophage infection of E. coli cells deficient in the enzyme tRNA nucleotidyltransferase (cca mutants) resulted in greatly decreased production of viable progeny phage compared to wild type cells. This decrease amounted to as much as 90% in the case of T-even bacteriophages, and 50-65% for T-odd bacteriophages. However, infection by the RNA phages, Qbeta and f2, was unaffected by the cca mutation. Examination of T4 infection of cca hosts indicated that phage development proceeded normally, that near-normal numbers of progeny particles were formed, but that most of these particles were non-viable. Possible functions for E. coli tRNA nucleotidyltransferase during bacteriophage infection are discussed.     

Temperate phages TP901-1 and phiLC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp. cremoris 3107

Five mutants of Lactococcus lactis subsp. cremoris 3107 resistant to phage TP901-1 were obtained after treatment with ethyl methanesulfonate. Two of the mutants were also resistant to phage phiLC3. The remaining three mutants were as sensitive as 3107. Mutants E46 and E100 did not adsorb the two phages. Mutants E119, E121 and E126 adsorbed phage phiLC3 as well as 3107 but phage TP901-1 with significantly reduced efficiency. All, except E46, could be lysogenized with phage TP901-BC1034, a derivative of TP901-1 harboring an erythromycin-resistance marker. However, the lysogenization frequency was 10(3)-10(4) fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutants. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle during infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901-1. As such impairment was not seen when infecting E119, E121 and E126 with phiLC3, we conclude that TP901-1 and phiLC3 either are differently triggered by their receptor or utilize different pathways of injection.     

A FhuA mutant of Escherichia coli is infected by phage T1-independent of TonB

Infection of Escherichia coli K-12 by phages T1 and phi 80 requires the FhuA outer membrane protein and the TonB protein. Mutations in the N-terminal globular domain close to the predicted channel in the beta-barrel of FhuA were created. The FhuA Delta 107-111 N104K K110D L111P mutant and the FhuA(L(109)DPNGLK(110)) insertion mutant were sensitive to phage T1, but nearly resistant to phage phi 80. FhuA Delta 107-111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants. The FhuA mutants showed no altered sensitivity to phage T5. Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities. In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages.

A method was developed for the selective enumeration of F male-specific bacteriophages in samples of environmental waters. The host strain for the phages, Escherichia coli HS(pFamp)R, has three antibiotic resistance markers, ampicillin on the Famp plasmid, which codes for pilus production, and streptomycin and nalidixic acid on the chromosome. The strain is resistant to coliphages T2 to T7 and phi X174. More than 95% of the phages from environmental samples which plaqued on the host strain were F male specific. The host bacterium had a higher plaquing efficiency than E. coli K-12 Hfr for F-specific phages in stock suspensions and sewage effluents. The F male-specific phage levels in prechlorinated, secondary-treated sewage effluents generally were about 10(3) to 10(4) PFU/100 ml. The levels in the influents to the sewage treatment plants and in septic tank contents were about 10(5) PFU/100 ml. RNA-containing phages composed about 90% of the total F-specific phage population in sewage effluents.     

Transmission of F'lac plasmid from Escherichia coli K-12 to bacteria of the genus Erwinia]

The F'lac plasmid was transferred by conjugation from Escherichia coli K-12 W1655 to 21 lac- strains of Erwinia spp. (5.2 . 10(-6) to 6.8 . 10(-2) lac+ exconjugants per donor cell). Erw. herbicola and Erw. chrysanthemi were the better recipients than others. The degree of the stability of lac+ genes in Erwinia exconjugants depends on the strains. Stable exconjugants of Erwinia, which harbored F'lac plasmid, were able to utilize lactose, to transfer lac genes by conjugation to Erwinia spp. and E. coli, and were sensitive to the F-specific phages f1, f2, Qbeta. The F'lac plasmid was eliminated from the exconjugants by the treatment with acridine orange, which indicates that this genetic element is not integrated into the Erwinia chromosome.     

Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qbeta) were exposed to UV radiation from 0 to 150 mJ.cm-2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qbeta having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ.cm-2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ.cm-2 and 60 mJ.cm-2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.     

Phages for the gliding bacteriumCytophaga johnsonae that infect only motile cells

Twenty-eight phages active againstCytophaga johnsonae have been isolated and placed into 16 groups based on phage size and morphology and on host range studies using a variety of mutants derived fromC. johnsonae. Several lines of evidence support the idea that these phages infect only actively motile cells: (i) many mutants selected for resistance to one phage are nonmotile and are resistant to all phages, (ii) nonmotile mutants, selected for their inability to spread on plates, are resistant to all phages, (iii) when nonmotile mutants revert to the motile condition, they regain sensitivity to some or all phages, and (iv) carbony-cyanidem-chlorophenylhydrazone inhibits motility and prevents adsorption of a test phage.