Regulation of acetohydroxy acid synthase activities in adenyl cyclase-deficient strains of Escherichia coli K-12

Previous findings suggested that cyclic AMP was involved in the regulation of ilvB(AHASI) only and that ilvG (AHASII) and ilvHI (AHASIII) were not controlled by this nucleotide. In this study, derepression patterns of total AHAS activities (ilvB and ilvHI) in adenyl cyclase-negative strains (i.e. cya-) were substantially reduced as contrasted with AHAS activity observed for cya+ strains. Further, the parental strains (cya+) consistently exhibited higher levels of AHAS activity than mutant strains (cya-) during carbon and energy downshifts. Other data suggested that the valine derepression signal could not override the necessity for cya gene product to yield maximal derepression of AHAS gene activities. Cyclic AMP stimulated AHAS gene activities under both in vivo and in vitro assay conditions. Thus, these data provide evidence for an absolute requirement of cAMP for maximal expression of the genes encoding for AHAS activities of E. coli K-12.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.     

Occurrence of the bacteriophage lambda receptor in some enterobacteriaceae.

In Escherichia coli K-12, the receptor for phage lambda is an outer membrane protein which inactivates the phage in vitro. Lambda receptor activity was found in extracts from all wild strains of E. coli tested, although most of them fail to support growth of the phage. In some cases this failure is due to a masking of the receptor in vivo, the bacteria being unable to adsorb the phage or to react with antireceptor antibodies. In other cases, adsorption does occur, and the nature of the block in phage growth was not investigated. Most Mal+ strains of Shigella have lambda receptor, whereas most Mal- strains do not have it. Synthesis of the lambda receptor in Shigella is thus presumably controlled by the positive regulator gene of the maltose regulon as is the case in E. coli K-12. Phage lambda adsorbs on many Mal+ strains of Shigella and even yields plaques on some of them, although at a low frequency. No lambda receptor activity could be found in extracts of several strains of Salmonella and Levinea.     

Recombination of bacteriophage lambda in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.     

A grpE mutant of Escherichia coli is more resistant to heat than the wild-type

The grpE gene of Escherichia coli is essential for bacteriophage lambda DNA replication and is also necessary for host RNA and DNA synthesis at high temperature. A grpE mutant of E. coli was found to be substantially more resistant to 50 degrees C heat treatment than the wild-type. Upon receiving a 42 degrees C heat shock for 15 min, both the wild-type and the grpE mutant became more resistant to heat (i.e. they became thermotolerant). A grpE+ revertant behaved similarly to the wild-type in that it was more sensitive to heat than grpE cells. In addition, grpE cells had the same H2O2 and UV sensitivity as the wild-type. This implies that the conditions for which a grpE mutation is beneficial are unique to heat exposure and are not caused by H2O2 or UV exposure. Furthermore, synthesis of heat-shock proteins occurred sooner in the grpE mutant than in the wild-type, indicating that the grpE gene of E. coli may influence the regulation of the heat-shock response.     

Bacteriophage infection is targeted to cellular poles

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Novel selection for tetracycline- or chloramphenicol-sensitive Escherichia coli.

A method for selecting tetracycline- or chloramphenicol-sensitive Escherichia coli cells from a population of predominantly resistant cells is described. This method depends on the inability of drug-sensitive cells to induce lambda receptors in the presence of chloramphenicol or tetracycline, protein synthesis-inhibiting drugs. The addition of bacteriophage lambda vir to a mixture of drug-sensitive and drug-resistant cells, induced for lambda receptors in the presence of tetracycline or chloramphenicol, preferentially kills the drug-resistant cells (which are capable of inducing lambda receptors). The result is a culture enriched for the sensitive cells. Several common strains used for transformation were compared for their ability to be selected. E. coli 294 was found to be superior.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Identification of a second tetracycline-inducible polypeptide encoded by Tn10.

Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10. One of these polypeptides was the previously identified 36,000-dalton TET polypeptide. The other two had approximate sizes of 25,000 and 13,000 daltons. The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts. Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts. However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts. An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides. The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E. coli strains carrying the amber suppressors supD, supE, and supF. In general, strains containing these nonsense suppressors were less resistant to tetracycline.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

Effect of cyclic 3',5'-adenosine monophosphate on the growth rate of Escherichia coli

Cyclic 3',5'-adenosine monophosphate (cAMP) inhibits the rate of Escherichia coli growth in media with glucose. When the exogenous nucleotide is added, the generation time and the lag phase become longer. These parameters decrease if cAMP is entirely absent from the cya- mutant as compared to the parent cya+ strain. The nucleotide exerts a low activity in media with glycerol. The action of cAMP is highly specific.