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PubMed2000

Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C.

Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes. The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth. The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C. A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein. In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system. Furthermore, the interaction between C and E, when analysed in the yeast two-hybrid system, is blocked by the sos mutation. An analysis of C mutants indicates that the dimerization function of C is located in the C-terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA. The sos mutation does not affect the capacity of the protein to dimerize. Using the yeast two-hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor. However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild-type C repressor.

Genes, Viral↗
PubMed2000

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Bacillus subtilis↗
PubMed2004

Genome-wide molecular dissection of serotype M3 group A Streptococcus strains causing two epidemics of invasive infections.

Molecular factors that contribute to the emergence of new virulent bacterial subclones and epidemics are poorly understood. We hypothesized that analysis of a population-based strain sample of serotype M3 group A Streptococcus (GAS) recovered from patients with invasive infection by using genome-wide investigative methods would provide new insight into this fundamental infectious disease problem. Serotype M3 GAS strains (n = 255) cultured from patients in Ontario, Canada, over 11 years and representing two distinct infection peaks were studied. Genetic diversity was indexed by pulsed-field gel electrophoresis, DNA-DNA microarray, whole-genome PCR scanning, prophage genotyping, targeted gene sequencing, and single-nucleotide polymorphism genotyping. All variation in gene content was attributable to acquisition or loss of prophages, a molecular process that generated unique combinations of proven or putative virulence genes. Distinct serotype M3 genotypes experienced rapid population expansion and caused infections that differed significantly in character and severity. Molecular genetic analysis, combined with immunologic studies, implicated a 4-aa duplication in the extreme N terminus of M protein as a factor contributing to an epidemic wave of serotype M3 invasive infections. This finding has implications for GAS vaccine research. Genome-wide analysis of population-based strain samples cultured from clinically well defined patients is crucial for understanding the molecular events underlying bacterial epidemics.

Antigens, Bacterial↗
PubMed2005

Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response.

Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.

Animals↗
PubMed2005

Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.

Identification of the genetic events that contribute to host-pathogen interactions is important for understanding the natural history of infectious diseases and developing therapeutics. Transcriptome studies conducted on pathogens have been central to this goal in recent years. However, most of these investigations have focused on specific end points or disease phases, rather than analysis of the entire time course of infection. To gain a more complete understanding of how bacterial gene expression changes over time in a primate host, the transcriptome of group A Streptococcus (GAS) was analyzed during an 86-day infection protocol in 20 cynomolgus macaques with experimental pharyngitis. The study used 260 custom Affymetrix (Santa Clara, CA) chips, and data were confirmed by TaqMan analysis. Colonization, acute, and asymptomatic phases of disease were identified. Successful colonization and severe inflammation were significantly correlated with an early onset of superantigen gene expression. The differential expression of two-component regulators covR and spy0680 (M1_spy0874) was significantly associated with GAS colony-forming units, inflammation, and phases of disease. Prophage virulence gene expression and prophage induction occurred predominantly during high pathogen cell densities and acute inflammation. We discovered that temporal changes in the GAS transcriptome were integrally linked to the phase of clinical disease and host-defense response. Knowledge of the gene expression patterns characterizing each phase of pathogen-host interaction provides avenues for targeted investigation of proven and putative virulence factors and genes of unknown function and will assist vaccine research.

Animals↗
PubMed2001

Epitope tagging of chromosomal genes in Salmonella.

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640-6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage lambda red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Amino Acid Sequence↗
PubMed1970

Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2.

The efficiency of plating of wild-type lambda on a host lysogenic for P2 is less than 10(-6), and only a small number of infected cells produce progeny phage. Lambda can adsorb and inject its DNA normally in such cells; the DNA can circularize and is not nicked or degraded, but replication is severely impaired. Mutants of P2, which as prophages no longer interfere with lambda, have been isolated and found to be recessive to wild type, implying that P2 prophage codes for a diffusible product involved in lambda interference. The P2 gene product responsible for preventing lambda growth also kills recombination-deficient bacteria of the recB and recC classes under conditions where P2 does not normally kill the host. Mutants of lambda that are resistant to interference are recessive to wild-type lambda. Thus lambda actively participates in its own interference. The lambda-mutants that are resistant to interference are unable to synthesize at least two nonessential proteins. In addition, they are unable to grow on recombination-deficient bacteria of the recA class, but they can grow on recA recB double mutants.

Carbon Isotopes↗
PubMed1972

Positive control of endolysin synthesis in vitro by the gene N protein of phage lambda.

Positive control in vitro by gene N protein of bacteriophage lambda was demonstrated. lambda DNA was used to direct in vitro synthesis of lambda endolysin in a cell-free protein-synthesizing preparation derived from Escherichia coli. The endolysin synthesis depends on the concomitant in vitro synthesis of lambda gene N protein. When lambda N(-) DNA was used to direct the cell-free preparation, endolysin was made only if extract was added from cells in which a lambda prophage had been induced. The use of various prophage deletion strains proved that if this stimulating activity made in vivo is coded by a known lambda gene, it must be coded by gene N. The ability to stimulate endolysin synthesis in vitro on a lambda N(-) DNA template, therefore, constitutes an assay for N protein.

Chloramphenicol↗
PubMed1974

Interaction of bacterial and lambda phage recombination systems in the x-ray sensitivity of Escherichia coli K-12.

E. coli cells lysogenic for the thermoinducible prophage lambdacI857 can be transiently induced by a brief heat treatment. Although this treatment does not kill the cells, some lambda products normally formed during vegetative phage development are made that can alter the response of host cells to x-irradiation by causing an increase in radioresistance. This increased resistance is particularly striking in the recombination-deficient recB-strain, which is normally much more radiosensitive than its recB(+) parent. After pulse-heating at 42 degrees , the survival curve of E. coli recB(-) lysogenized with lambdacI857 does not differ from that of the wild-type strain. Since lambda red mutants do not increase the radioresistance of recB(-) strains, both lambda red gene products, lambda exonuclease and beta-protein, are required to compensate for the missing recB product. Furthermore, phage-induced radioresistance also occurs in recB(+) lysogens even when they carry lambda red(-), but not when the lambda prophage is gam(-). Thus, in wild-type cells, phage-induced radioresistance requires some interaction between the bacterial recB gene product (exonuclease V) and the phage lambda-protein.

Coliphages↗
PubMed1974

Thermal enhancement of ultraviolet mutability in a tif-1 uvrA derivative of Escherichia coli B-r: evidence that ultraviolet mutagenesis depends upon an inducible function.

Inhibition of DNA replication in E. coli results in coordinate expression of a cluster of diverse functions, including prophage induction and filamentous growth, many of which are thermally inducible, without inhibition of DNA synthesis, in strains carrying a mutation in the "tif" locus. It has been proposed that one of the inducible functions in this cluster is responsible for the ultraviolet mutability of E. coli. As a test of this hypothesis, the effect of postirradiation temperature elevation on ultraviolet mutability was compared in strains differing by a tif mutation. In a tif(+) strain, raising the temperature to 42 degrees after irradiation did not change the frequency of induced mutations to tryptophan-independence. Similar treatment of a tif-1 strain resulted in as much as a 10-fold increase in the induced mutation yield. Thermal enhancement of ultraviolet mutability in the tif-1 strain was prevented or promoted by agents known to exert parallel effects on thermal induction of lambda prophage in tif lysogens. The results support the hypothesis that ultraviolet mutability in E. coli depends upon an inducible function that is normally expressed only when DNA replication is inhibited.

Adenine↗
PubMed1974

Release of polarity in Escherichia coli by gene N of phage lambda: termination and antitermination of transcription.

The induction of lambda prophage provokes the constitutive expression of the adjacent gal operon in E. coli. This "escape synthesis" can result from transcription that initiates at a phage promoter and extends into the gal operon. The effect requires the product of the lambda gene N. N-mediated transcription not only fails to terminate at the prophage-bacterial junction and at the ends of bacterial operons, but ignores termination signals caused by polar insertions or ochre mutations within gal. Suppression of polarity by N-function is a cis-effect; only transcription initiated at the phage promoter is influenced. We propose that the transcription complex is influenced by N-product to become termination-resistant at a site in the phage genome (juggernaut model). This site appears to be at or near the phage promoter.

Chromosome Mapping↗
PubMed1975

Acquisition of a determinant for chloramphenicol resistance by coliphage lambda.

A determinanat for chloramphenicol resistance, cam, initially detected on a resistance transfer factor (RTF) and since transferred to phage P1, may be acquired from P1 by coliphage lambda. Lambdapcam are obtained when a lambda prophage is induced in bacteria which also harbor P1 cam prophage. Lambdacam formation is not dependent upon host Rec or lambda Red recombination functions. Electron microscopic heteroduplex analysis shows that the cam locus in two lambdapcams is a 5% addition of DNA in the b2 region of lambda, not contiguous with att. The extent and nucloetide sequence of the DNA insertion in the two independent lambdapcam isolates appear to be the same though they are located at different sites within the b2 region. We conclude that the determinant for chloramphenicol resistance is contained on a unique piece of DNA which facilitates its insertion into a number of unrelated genomes.

Chloramphenicol↗
PubMed1976

Induction of sigma factor synthesis in Escherichia coli by the N gene product of bacteriophage lambda.

Thermoinduction of cells of E. coli carrying prophage lambdacI857 within the bfe gene brings about not only "escape synthesis" of core subunits of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2-7-7-6), but also a striking stimulation of sigma factor synthesis. The latter phenomenon, termed sigma induction, is generally observed after lambda phage infection or prophage induction. A series of experiments with various bacterial and phage strains led us to conclude that the N gene product of lambda is directly involved to the sigma induction. These and other results obtained with mutants defective in transcription termination factor rho suggest the involvement of a rho-sensitive site in the control of sigma gene expression in E. coli.

Chromosome Mapping↗
PubMed1976

Fusion of bacterial protoplasts.

Prototrophic Bacillus subtilis cells can be formed in the presence of DNase as a result of cell fusion occurring in mixed populations of protoplasts derived from two parental strains which are both nutritionally-complementing and polyauxotrophic. No prototrophs ever appear from mixed nonprotoplasted bacteria, or from the auxotrophic parental protoplasts plated separately. The frequency of prototroph formation, which is appreciable only when the mixed protoplasts are exposed to polyethylene glycol treatment, may exceed 1 X 10(-4) of the total protoplast population initially present, which is 1 to 4 X 10(-3) of those protoplasts which reverted to the bacillary form. It is strongly dependent on the number and chromosomal location of the markers used in the selection of the prototrophs, and it is unaffected when either one of the parental strains bears the phage phi105 in the inducible prophage state. No auxotrophic bacteria, parental or otherwise, were found as segregants from repeatedly isolated protrotrophic clones growing in a nonselective medium. Unselected markers segregate among the selected recombinants. It is concluded that the observed formation of prototropic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.

Bacillus subtilis↗
PubMed1978

Early events and mechanisms in the induction of bacterial SOS functions: analysis of the phage repressor inactivation process in vivo.

Different inducing agents and treatments produced distinctly different kinetic patterns of inactivation of prophage repressor molecules. The different patterns were related to differences in the initial altered states of DNA that were produced. The timing of appearance of DNA degradation was correlated with the time needed for repressor inactivation. These characteristics suggest that all the inducing treatments lead to the formation of a final predegradative DNA structure(s) (probably involving scissions) that is acted on by specific DNases, including the recBC DNase, to produce the signals for the induction of prophage.

Bleomycin↗
PubMed1980

Inversions of specific DNA segments in flagellar phase variation of Salmonella and inversion systems of bacteriophages P1 and Mu.

Prophages P1 and Mu produces a trans-acting factor possessing the din+ activity which catalyzes the inversion of the specific DNA segment responsible for flagellar phase variation of Salmonella, din mutants were isolated from PICMclr100 phage by selecting phages that did not suppress the yh2 mutation of Salmonella in prophage state. No inversion loop structure was detected among DNA forms arising after denaturation and rehybridization of DNAs extracted from the din mutants. The DNA fragment containing C region of P1 was cloned on a plasmid vector, pCR1. The resulting hybrid plasmid, pKK2, was shown to possess the din+ activity: the vh2 mutant of Salmonella harboring the plasmid changed the flagellar phase. From analysis of the plasmid by use of BamHI and Bgl II, the din gene specifying the din+ activity was located near or within the C region of P1. It is highly plausible that the din gene of P1 was also involved in the inversion of the C region. Similarly, the DNA fragment containing the G and beta segments of Mu was cloned on pCR1. The resulting hybrid plasmid, pII101, also possessed the din+ activity.

Bacterial Proteins↗
PubMed1980

DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli.

Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector [Casadaban, M.J. & Cohen, S.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4530-4533] to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

DNA Repair↗
PubMed1981

Retroregulation of the int gene of bacteriophage lambda: control of translation completion.

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.

Bacteriophage lambda↗