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Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phagehost interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniae. [ABSTRACT FROM AUTHOR]

Bacteria have developed various strategies for phage resistance. Infection with phage induces the transcription of part of the phage resistance gene, but the regulatory mechanisms of such transcription remain largely unknown. The phage resistance gene nonA is located on the SPβ prophage region of the Bacillus subtilis Marburg strain genome. The nonA transcript was detected at the late stage of SP10 infection but is undetectable in noninfected cells. The nonA transcript was detected after the induction of the sigma factor Orf199-Orf200 (σOrf199-200), when sigma factors encoded in the SP10 genome were expressed from a xylose-inducible plasmid. Thus, the SP10 sigma factor is an activator of a set of SP10 genes and nonA. The nonA gene encodes a 72-amino-acid protein with a transmembrane motif and has no significant homology with any protein in any database. NonA overexpression halted cell growth and reduced the efficiency of B. subtilis colony formation and respiration activity. In addition, SP10 virion protein synthesis was inhibited in the nonA+ strain, and SP10 virion particles were scarce in it. These results indicate that NonA is a novel protein that can abort SP10 infection, and its transcription was regulated by SP10 sigma factor. [ABSTRACT FROM AUTHOR]

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium .

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lanibdoid phages is mostly limited to the capsid genes. Lula/phi8O's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80's lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80's SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi8o, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion. [ABSTRACT FROM AUTHOR]

Studies dual regulatory control of particle maturation function of bacteriophage P1 in Escherichia coli. Arrangement of promoter elements upstream the bacteriophage P1 maturation gene mat; Determination of the nucleotide sequence of mat; Complexity of the P1 regulatory cascade; Control of mat expression by a hybrid promoter; Ability of P1 to fine-tune the expression of its genetic information.

Examines the functional and genetic expression of CTX lysogenic bacteriophage genomes in Vibrio cholerae. Sites of CTX prophage integration; Structures of CTX prophages; Failure of prophage arrangements to produce extrachromosomal CTX DNA; Differences between phage genomes; Generation of proteins necessary for CTX production.

Investigates the function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal. Manner of cleavage of the Lys44 in Escherichia coli; Dependence of lytic activity in oenococcal cells on Lys44 processing; Progression of synthesis and export of Lys44 in the infected host.

Examines the phage-encoded proteins involved in site-specific excision of the temperate lactococcal bacteriophage TP901-1. Integration of the genomes site into the bacterial host chromosome; Recombination between the attachment sites attB and attP on the bacterial and phage genomes; Establishment of the lactococcal strain containing the excision vector.

Examines the interaction of T4 bacteriophage-encoded anti-sigma factor, asiA, and Escherichia coli with the use of a two-hybrid system. Identification of the minimum region involved in the interaction by sigma70 truncation; Implications of the increased affinity of truncated sigma70 fragments to asiA; Evaluation of the coexpression system.

Investigates nested genes kil and eta in Escherichia coli bacteriophage P4. Effect of the phage P4 to immunity-specific transcription termination; Transcription of the left operon; Constitutive and regulated P... promoter; Rho-dependent termination site; Effect of overexpression of kil and eta; Coding sequences; Ash- phenotype.

Studies the bacteriophages with genomes of segmented double-stranded RNA. isolation of eight bacteriophages; Attachment of most isolates to the outer membrane; Establishment of carrier states by viruses attaching directly to the outer membrane; Constituents of three segments of double-stranded RNA in isolates; Similarity of genomic-segment sizes.

Investigates whether site-specific recombination of bacteriophage P22 does not require integration host factor. Sequences of synthetic oligonucleotides for amplification of P22 integase attachment sites and cloning of int genes; Frequencies of lysogeny by bacteriphages P22 and P22goh5.

Investigates the nucleotide sequence of the major control region of Vibrio cholerae bacteriophage K139. Relationship between K139 and pathogenic O1 Vibrio cholerae strains; Similarity of K139 to P2 bacteriophage family; Integration of K139 between flagellin genes flaA and flaC of O1 El Tor and O139 Vibrio cholerae lysogenic strains.

Describes the genetic basis of adsorption in the filamentous phage phiLf gene III infecting the gram negative bacteria Xanthomonas campestris. Coding region of the phiLf gene III; Sequence analysis of the gene; Determinants of the host specificity of the phiLf gene III.

Analyzes the structural determinants of three functions of gp33: binding to RNA polymerase core, mediation of transcriptional enhancement by the DNA polymerase accessory proteins, and inhibition of unenhanced transcription. Site-directed mutagenesis; Overexpression and 35S labeling of gp33 mutant proteins; Single round transcription.

Characterizes the terminators of the lys and P transcripts to further understanding of the Mu late operon structure and factors which may influence transcription termination. Determination of the lengths of the two transcripts; Localization of the 3' ends of the lys and P transcripts.

Characterizes the temperate lactococcal phage TP901-1 as induced by ultraviolet light from Lactococcus lactis subsp. cremoris 901-1. Restriction map of phage TP901-1; Identification and DNA sequences of attPk, attL and attR; Lysogenic life cycle of the phage; Cloning of TP901-1 DNA; Construction of an integration vector; Cloning of the attL and attR regions.

Discusses the genetic encoding of lysogenic bacteriophages Shiga toxin 2 from enterohemorrhagic Escherichia coli O157:H7 Shiga toxin. Toxin encoding in various E. coli strains; Morphology of the 933W virion; Identification of the virion DNA endpoints; Nucleotide sequencing promoters, operators and terminators.

Studies the oligohistidine tag mutagenesis of the lambda holin gene. Four lysis genes of the bacteriophage lambda; Materials and methods used in the study; Data collection and analysis; Results.

Describes the cloning and analysis of the endolysin genes from different Bacillus cereus bacteriophages. Presentation of an evidence for the relationship of the phage enzymes to autolysins from other bacilli; Determination of the substrate bonds cleaved in Bacillus cereus cell walls; Contentions that Ply21 genes may be endowed with a signal peptide.

Demonstrates the role of T4 RNase HI or Escherichia coli DNA polymerase I in phage replication. Independence of wild-type T4 phage production from polA12 control; Reduction of phage production with the deletion of C-terminal amino acids from T4 RNase H; Restoration of phage production in the nonpermissive host by providing the T4 rnh gene on a plasmid.

Determines the minimal segment of DNA required for attB function in Mycobacterium smegmatis. Identification of strand exchange for integrase-mediated recombination between Mycobacterium smegmatis and Mycobacteriophage L5.

Describes the cloning and sequencing of bacteriophage P1virS genes 17, lydA and lydB with their respective alleles. Construction of a lysis cassette; Mapping of the LPr21 late promoter sequence; Mapping of gene 17.

Investigates why gene 19 of bacterial plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection. Construction for plasmid R1-16 with mutations in gene 19; Pilus synthesis and assembly; Kinetics of the DNA transfer; Surface exclusion; Number of viable cells; Phage RNA replication.

Examines the penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells. Measurement of Dam methylation of the entering genome; Assays of phage genome entry; Prevention of total methylation of the phage genome during infection of F-containing cells.

Examines the rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli. Assays of phage genome entry into cells; Analysis of protein synthesis during T7 infection; Methylation of T7 DNA.

Investigates the lactococcal AbiD1 gene coding for phage abortive infection. Activity of abiD1 determined by plasmid pIL105 on prolate- and small-isometric-head phages of the C6A and 936 phage groups; Determination of Abi phenotype by a single gene designated abiD1.

Describes the purification of the bacteriophage P4 delta protein. Use of nitrocellulose filtration and gel mobility shift assays; Characterization of P4 delta protein as a transcriptional activator of the late genes of P4 as well as the late genes of its helpers.

Examines S gene expression and the timing of lysis by bacteriophage gamma. Kinetics of S mRNA synthesis; Kinetics of S protein accumulation during gamma vegetative cycle; Comparison of S protein accumulation in mutants with altered lysis timing; Structural features.

Describes the identification of related genes in bacteriophages phi80 and P22 whose products are inhibitory for phage growth in Escherichia coli IHF mutants. Marker rescue to localize rha gene; Sequence of gene and allele; Identification of functional homolog; Production of Rha during infection.

Investigates the superinfection exclusion gene (sieA) of bacteriophage P22. Identification and overexpression; Localization of gene product; Mapping by subcloning experiments; Nucleotide sequence of region; Determination of translational start.

Studies the effect of streptomycin- and rifampin-resistant mutants of Escherichia coli on F exclusion of bacteriophage T7. Synthesis of F plasmid protein PifA; Variation in severity of F exclusion; RpsL gene mutations.

Studies the role of bacteriophage P2 ogr gene in the positive regulation of transcription from P2 late promoters. Effects of other chromosomal alleles; Leucine residues; Interaction between ogr and alpha subunit of ribonucleic acid (RNA) polymerase.

Discusses the functions involved in bacteriophage P2-induced host cell lysis and the identification of a tail gene. Strains; Plasmids; Growth conditions; DNA manipulations and analysis; Synthetic oligonucleotides; Lysozyme assay; Cloning and sequence determination of lysis region; Gene K; Lysis phenotypes of amber mutants; Activities of lysis proteins.

Discusses the interdependence of the c4, icd and ant genes in the P1 immI operon as revealed by second-site suppressors of the bacteriophage P1 virs mutant. Encoding of an antisense RNA; Inhibition of synthesis of an antirepressor by acting on a target ant messenger RNA; Implications on transcription, coupling, lethality and synthesis regulation.

Discusses the cloning and physical mapping of the early promoters of bacteriophage D3 of Pseudomonas aeruginosa. Determination of relative strengths, direction of transcription and regulation by repressor; Promoter activity; Operator-promoter complexes; Analogy to the immunity region of coliphage lambda; Location and effect of insertion element IS222.

Describes the plasmid expression system of the lipid -containing membrane of bacteriophage phi6. Protein composition of the membrane; Relationship between membrane protein P9 and morphogenetic protein P12; Demonstration of the presence of protein P9 through immunoblotting.

Studies the functional organization of the bacteriophage PRD1 genome. Replication through a protein-primed mechanism; Chloramphenicol acetyltransferase; Identification of promoter regions controlling PRD1 transcription.

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to L lysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by L overcomes dnaJ (Lodj) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ~20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of φX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. [ABSTRACT FROM AUTHOR]

The filamentous bacteriophage CTXΦ transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTXΦ genes required for virion production initiate transcription from the strong PA promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, PR, and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of PR transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position -23.5, within the PR spacer region. Our results indicate that LexA, when bound to its single site in the CTXΦ prophage, both represses transcription from PA and coactivates transcription from the divergent PR. We propose that LexA coordinates PA and PR prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTXΦ lysogens. [ABSTRACT FROM AUTHOR]