Early development of bacteriophages SP01 and SP82G in minicells of Bacillus subtilis.

SP01- and SP82G-infected Bacillus subtilis CU403 divIVBI minicells synthesize 13 easily detectable early RNA species with molecular weights ranging from 60 × 10³ to 430 × 10³. Comparison of in vivo and in vitro translation of early messenger RNA indicates that five early mRNAs of SP01 are synthesized but not translated unless protein synthesis has been permitted in the infected minicell, providing evidence for a translation control mechanism. A sequential appearance of 48 polypeptides has been determined in SP01-infected minicells. The polypeptides have been grouped into two classes of early polypeptides, i.e. those encoded by early mRNA and three subsequent classes as demonstrated by the analysis of polypeptides synthesized in minicells infected with the SP01 mutants, susF21, susF4 and susF14. Phage capsid proteins are not synthesized in minicells. RNA synthesized in infected minicells is subject to turnover. The individual mRNA species have differing functional stabilities ranging from a loss of only 50% functional activity, in 20 minutes at 37 °C, to loss of over 99% activity.Infection of anucleate minicells has been shown to be a very simple method for comparison of closely related phages (slight differences are detected between SP01- and SP82G-encoded mRNA and polypeptides), detection of polypeptides affected by amber mutations and the analysis of early events in phage development in the absence of host syntheses.     

Control of lysogeny and immunity of Bacillus subtilis temperate bacteriophage SP beta by its d gene.

The d gene from the Bacillus subtilis temperate bacteriophage SP beta was isolated. When introduced into an SP beta-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP beta phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SP beta phage genome. The resulting SP beta phage grew lytically and formed clear plaques on sensitive bacteria. Although the cloned d gene confers immunity to the host, we could not detect complementation of the d gene by mixed infection with SP beta d2 and various SP beta c mutants. The nucleotide sequence of the 1,033-base-pair PstI-to-EcoRI fragment containing the d gene was determined; it includes an open reading frame that could potentially encode a protein of 227 amino acids. The gene was mapped within the PstI H fragment on the phage genome, which positions the d gene about 25 kilobases from the right end of the phage genome. It is transcribed from right to left.     

DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01

SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M. Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene. Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation. We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A.     

Complete genomic sequence of the virulent Salmonella bacteriophage SP6

We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.     

Site-specific DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein

The bacteriophage SP01 genome encodes a virus-specific type II DNA-binding protein, TF1. The bacterial proteins of this ubiquitous and evolutionarily conserved class are thought to bind non-specifically to DNA. In contrast, the experiments described here demonstrate that TF1 binds to specific sites in SP01 DNA. Several of these sites have been characterized by DNase I 'footprinting' and four of them have been shown to overlap strong phage promoters for Bacillus subtilis RNA polymerase holoenzyme. We speculate on the possible structural basis of site-selective DNA binding by a protein of this class.     

Effects of the Decay of Incorporated Radioactive Phosphorus on the Transfer of the Bacteriophage SP82G Genome

The last genetic markers to be transferred during bacteriophage SP82G infection have a higher sensitivity to the decay of incorporated radioactive phosphorous (³²P) than those which are located on the proximal end of the genome. If ³²P decay is permitted to take place after DNA transfer is complete (in frozen infective centers) and in the absence of DNA replication, no dependence of marker sensitivity on map position is observed. These results indicate that the decay of incorporated ³²P leads to damages that prevent the efficient transfer of portions of the genome distal to the lesion. At 4 C, failure to transfer some portion of the genome occurs in 49% of all lethal events. Even though damages that prevent transfer of the genome are in themselves lethal, they do not prevent rescue of genetic markers on portions of the genome that are transferred. The portion of the genome that is transferred, is transferred at the same rate as an undamaged genome. We interpret these results to mean that double-strand breaks in the DNA are the lesions that prevent distal transfer and that single-strand breaks have little or no effect on the transfer of the bacteriophage SP82G genome.     

Isolation and characterization of a bacteriophage forBacillus licheniformis A5

The isolation of a virulent bacteriophage forBacillus licheniformis A5 is reported. This bacteriophage, designated NLP-1, has an icosahedral head 100 nm in diameter and a contractible tail with a maximum length of 130 nm. Its DNA has a density of 1.741 g/cm³ and aT _m of 78.4°C. Base composition analysis showed that thymine is absent and is replaced by hydroxymethyluracil. NLP-1 appears to belong to theBacillus group of bacteriophages that includes SP01 and SP82. It will infectB. cereus T andB. brevis 8185, but will not infectB. subtilis W23 or 168.     

Interference of plasmid pCM194 with lysogeny of bacteriophage SP02 in Bacillus subtilis.

Three observations indicated that the 2-megadalton chloramphenicol resistance plasmid pCM194 interferes with SP02 lysogeny of Bacillus subtilis. SP02 plaques formed on B. subtilis(pCM194) appeared almost clear, whereas plaques produced on plasmid-free or pUB110-containing cells contained large turbid centers. The number of phages spontaneously liberated by B. subtilis(SP02) was increased 10-fold or more when pCM194 was also present in the lysogens. Lastly, growth of B. subtilis(SP02, pCM194) for approximately 20 to 25 generations resulted in essentially complete loss of the prophage. This interference was not observed with pUB110 or pE194, and the pCM194 interference was not directed against B. subtilis temperate phage phi 105, which is unrelated to SP02. Lytic replication of SP02 appeared to be unaffected by pCM194. pCM194 interference with SP02 lysogeny was demonstrable in recombination-proficient strains and a recE mutant of B. subtilis. SP02 prophage which were noninducible due to the phage ind mutation were resistant to pCM194 interference. pCM194 interference was lost when the entire pCM194 molecule was joined at its unique HpaII site or at one of the two MboI sites to pUB110 or pUB110 derivatives. pBR322 joined to pCM194 at the same MboI site or at the HindIII site produced chimeras that retained the ability to interfere with SP02 lysogeny. A three-part plasmid constructed by joining pBR322 to pCM194 (at HindIII sites) and to pE194 (at PstI sites) was compatible with the SP02 prophage and showed a temperature-sensitive replication phenotype characteristic of the pE194 replicon. One explanation for the interference involves competition for a host component between an SP02 genome attempting to establish lysogeny and plasmids whose replication is directed by the pCM194 replicon.     

The role of temperate bacteriophage SP beta in prophage-mediated interference in Bacillus subtilis.

Virulent bacteriophage phi 1 grows on a variety of Bacillus subtilis strains, mutants of this virus which abortively infect the transformable bacillus. B. subtilis 168, while retaining the ability to productively infect related bacteria have been found. In the present study, we demonstrate that the inability of one such variant, phi 1m, to develop normally in strain 168 is mediated by cryptic prophage SP beta. The latter is a temperate bacteriophage which is carried by B. subtilis 168 and most strains derived from this bacterium. Phi 1 m infection of SP beta lysogens begins with apparently normal adsorption, penetration, and inititaion of virus-directed syntheses. At about the 20th min of the latent period, however, there is an abrupt cessation of nucleic acid synthesis and cellular respiration, accompanied by a change in cell permeability. This course of events can be altered to a permissive infection by mutation in the mpi gene of SP beta, by mutation in the spoOA gene of the host, or by growing SP beta lysogens at high temperature. In addition, we found a second class of phi 1 mutants which abortively infect B. subtilis 168 derivatives even in the absence of the SP beta prophage.     

Comparison of abacavir-specific effector and proliferating functions of CD8 T cells in abacavir-treated HIV-1 patients

Susceptibility to abacavir hypersensitivity (ABH) in HIV-1-positive patients is strongly linked to the carriage of HLA-B*57:01 and the potential mechanism includes drug-specific activation of cytokine producing CD8 T cells exclusively in individuals carrying HLA-B*57:01. Here, we report a detailed characterization of abacavir-induced functional response of CD8 T cells in HLA-B*57:01^pos individuals. Peripheral blood mononuclear cells (PBMNCs) from HLA-B*57:01^posABH^pos and HLA-B*57:01^negABH^neg individuals were stimulated with abacavir. Multicolor flow cytometry was performed to assess the cytokine (IFNγ) production and degranulation (CD107a expression) after 6–18 hr culture and to enumerate proliferating CD4/CD8 T cells by culturing carboxyfluorescein diacetate succinimidyl ester-loaded PBMNCs for 7 days. CD8 T cells from HLA-B*57:01^posABH^pos individuals were multifunctional: proliferating, IFNγ producing, degranulating (CD107a^pos), and both degranulating and IFNγ producing (CD107a^posIFNγ^pos). Degranulating CD8 T cells in general and both degranulating and IFNγ producing CD8 T cells in particular dominated abacavir-specific immune response. All functional responses were partially blocked by addition of HLA-B*57:01-reactive Bw4 mAb, but not by non-HLA-B*57:01-reactive Bw6 mAb. In conclusion, the study demonstrates that abacavir-specific CD8 T-cell-restricted immune response in HLA-B*57:01^posABH^pos HIV-1 patients has multiple effector and proliferating functions, where the primary effector response appears to be the release of cytolytic granules. The findings have implications for immunotherapy of HLA-related drug hypersensitivities.     

Sequence of the bacteriophage SP01 gene 30.

The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.     

Ligation of highly modified bacteriophage DNA

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4′,5′-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, λ DNA to be ligated was as follows: $\text{λ}\text{DNA}\text{=}\text{XP}\text{12}\text{DNA}\text{>}\text{SP}\text{82}\text{DNA}\text{?}\text{nonglucosylated}\text{T}\text{4}\text{DNA}\text{>}\text{T}\text{4}\text{DNA}\text{=}\text{PBS}\text{1}\text{DNA}\text{?}\text{SP}\text{15}\text{DNA}$. TaqI-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and λ DNAs was equally efficient. We conclude that the poor ligation of TaqI fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.     

Genome Sequence of Enterobacter sp. Strain SP1, an Endophytic Nitrogen-Fixing Bacterium Isolated from Sugarcane

Enterobacter sp. strain SP1 is an endophytic nitrogen-fixing bacterium isolated from a sugarcane stem and can promote plant growth. The draft genome sequence of strain SP1 presented here will promote comparative genomic studies to determine the genetic background of interactions between endophytic enterobacteria and plants.     

Complete genome sequence of the cellulolytic planctomycete Telmatocola sphagniphila SP2T and characterization of the first cellulolytic enzyme from planctomycetes

Planctomycetes of the family Gemmataceae are strictly aerobic chemo-organotrophs that display a number of hydrolytic capabilities. A member of this family, Telmatocola sphagniphila SP2^T, is the first described planctomycete with experimentally proven ability for growth on cellulose. In this study, the complete genome sequence of strain SP2^T was obtained and the genome-encoded determinants of its cellulolytic potential were analyzed. The T. sphagniphila SP2^T genome was 6.59 Mb in size and contained over 5200 potential protein-coding genes. The search for enzymes that could be potentially involved in cellulose degradation identified a putative cellulase that contained a domain from the GH44 family of glycoside hydrolases. Homologous enzymes were also revealed in the genomes of two other Gemmataceae planctomycetes, Zavarzinella formosa A10^T and Tuwongella immobilis MBLW1^T. The gene encoding this predicted cellulase in strain SP2^T was expressed in E. coli and the hydrolytic activity of the recombinant enzyme was confirmed in tests with carboxymethyl cellulose but not with crystalline cellulose, xylan, mannan or laminarin. This is the first experimentally characterized cellulolytic enzyme from planctomycetes.     

An atypical deoxynucleoside triphosphate inBacillus subtilis infected with bacteriophage SP10c

In mature DNA ofBacillus subtilis phage SP10c, deoxythymidine monophosphate is partially replaced by a hypermodified nucleotide. During the interval of phage replication, infected cells contained greatly reduced levels of deoxythymidine triphosphate. However, an atypical mononucleotide, tentatively identified as 5-hydroxymethyldeoxyuridine triphosphate, was present during the interval of SP10c DNA synthesis. It is proposed that the atypical mononucleotide, and not deoxythymidine triphosphate, is a substrate for SP10c DNA replication.     

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.