Increased reproductive fitness of Escherichia coli lambda lysogens.

Lambda lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats. If the environment is changed to anaerobic growth, the situation is reversed, and the lysogen reproduces more slowly than the nonlysogen. Based on a tetrazolium dye assay, the increased fitness of the lambda lysogen during aerobic growth seems to result from a continued high metabolic rate as glucose becomes limiting, whereas the metabolic rate of the nonlysogen declines. The lambda rex gene is required for the growth advantage of lysogens since lack of rex function causes lambda lysogens to lose their reproductive advantage over nonlysogens.     

Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu.

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C. The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.     

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.     

P1 bacteriophage and tellurite sensitivity in Klebsiella pneumoniae and Escherichia coli

Klebsiella pneumoniae and Escherichia coli respond inversely toward P1 bacteriophage or TeO3(-2). Klebsiella pneumoniae is resistant to both antagonists and E. coli is sensitive. However, P1 cmts lysogens (P1 cmts resistant) of K. pneumoniae became sensitive to tellurite and when cured from P1 cmts regained resistance. Escherichia coli spontaneous mutants selected for resistance to either P1 or TeO3(-2) were collaterally resistant to the other. As well, TeO3(-3) enhanced the adsorption of P1 vir to both E. coli and K. pneumoniae. Several outer membrane proteins were enhanced in the K. pneumoniae lysogens and were reduced in E. coli lysogens; one of which was the same molecular weight (77 000) in both bacteria. When partially purified it enhanced the plaque efficiency of P1 vir. Lipopolysaccharide (LPS) from E. coli C600 inactivated P1 vir, but neither the P1 lysogens nor LPS derived from the lysogens inactivated P1 vir. Escherichia coli P1 lysogens produced only heptose-deficient LPS. It is suggested that both LPS and outer membrane protein(s) comprise the P1 receptor. TeO3(-2) may interact with one or both components.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

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.     

Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts.

Characterization of a putative intermediate in the Mu lysogenization pathway is possible in a variant Escherichia coli himD strain which exhibits greatly diminished lysogen formation. In this strain, most infecting Mu genomes form stable, transcribable, nonreplicating structures. Many of these genomes can be mobilized to form lysogens by a second Mu infection, which can be delayed by at least 100 min. This intermediate structure can be formed in the absence of Mu A or B function. We suggest that the inferred intermediate could be the previously reported protein-linked circular form of the Mu genome. Providing Mu B function from a plasmid enhances Mu lysogenization in this him strain, and the enhancement is much greater when both Mu A and B functions are provided.     

Maintenance of bacteriophage P1 plasmid.

Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.     

Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12.

Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms.     

Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system

Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.     

Virulent mutants of temperate phage Mu-1

Summary Virulent mutants of phage Mu have been isolated after mutagenesis. The virulent phenotype results from most probably 2 mutations located in the c-A region of the Mu genome.Vir mutants are trans-dominant; they induce the resident prophage upon infection in broth of any Mu lysogen. They however form plaques only on certain lysogens, that are monolysogenic for a mutant prophage. We further isolated secondary mutations in Mu Vir which suppress the virulent phenotype.     

Superinfection Exclusion by P22 Prophage and the Replication Complex

Wild-type sie(+) P22 prophage converted Salmonella typhimurium lysogens to exclude deoxyribonucleic acid (DNA) injected by superinfecting phage. DNA from a P22 superinfecting virulent phage associated with the replication complex in a sie(-) lysogen but not in a sie(+) lysogen.     

Lethal Transposition of Mud Phages in Rec(-) Strains of Salmonella Typhimurium

Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.     

Selection of laboratory wild-type phenotype from natural isolates of Escherichia coli in chemostats.

We have followed, in glucose-limited chemostats, the evolution of natural isolates of Escherichia coli possessing maximal growth rates of 0.48-1.43 doublings/h. Under these conditions a rapid-growth phenotype similar to that of standard laboratory wild-type strains was selected so that after 280 generations all of the cultures were characterized by bacteria with maximum growth rates close to 1.33 doublings/h. The growth yields of the natural isolates, on the other hand, were quite uniform and improved only slightly during the selection; it seems that the natural isolates are nearly maximally efficient at utilizing glucose. Some of the kinetic characteristics of ribosomes prepared from natural isolates vary markedly and in proportion to the growth rates of the original strains. After growth in glucose-limited chemostats, the ribosomes of all of the cultures become kinetically indistinguishable from those of laboratory wild-type bacteria. These observations confirm the interpretation that bacteria grown under normal laboratory conditions have been selected for maximum growth rates which demand maximum translation efficiency. In contrast, these characteristics do not seem to be strongly selected in the natural isolates.     

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.     

Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-phi 1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as phi CP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage phi CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some phi CP6-1 phage up to day 137, yet hardly any phi CP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.     

Inhibition of transformation in Streptococcus pneumoniae by lysogeny.

Streptococcus pneumoniae R6X was lysogenized with bacteriophage 304 isolated after mitomycin induction of an ungrouped alpha-hemolytic streptococcus. Lysogenized pneumococci lost their capacity to undergo genetic transformation: transformability was restored after cells were spontaneously cured of their prophage. Both lysogens and nonlysogens produced activator substance (competence factor), and both bound deoxyribonucleic acid in a deoxyribonuclease-resistant form. However, nonlysogens retained deoxyribonucleic acid after washing, whereas lysogens did not. The latter did not liberate phage nor (unlike nonlysogens) degrade transforming deoxyribonucleic acid and contained normal levels of endonuclease.     

Influence of iron-limited and replete continuous culture on the physiology and virulence of Neisseria gonorrhoeae

Neisseria gonorrhoeae strains P9-2 (PenS) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (micromax 0.1 h-1) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2- or 11-fold greater than during cystine- or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose- or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine- or glucose-limited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-O-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+O- gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (iron-replete) gonococci retained piliation but did not survive in the chambers. Transition from iron-limited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9, 3.7, 4.3 and 4.8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine- or glucose-limited bacteria.     

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by 相似文献