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Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

This chapter describes the procedure that we have used to introduce suppressible nonsense mutations into various genes of Bacillus subtilis bacteriophage SPO1. The targeted gene is cloned in a B. subtilis/Escherichia coli shuttle vector. Using an in vitro enzymatic procedure dependent on mutant oligonucleotide primers, a mutation is inserted into the cloned gene, replacing an early lysine codon (AAA or AAG) with a nonsense codon (TAG or TAA). The mutant plasmid is recovered by transformation into E. coli, and is then transformed into B. subtilis carrying a suppressor that inserts lysine at TAG or TAA codons. Recombination is allowed between the mutant plasmid and superinfecting wild-type SPO1, and mutant progeny phage are identified by plaque-lift hybridization to labeled oligonucleotides having the mutant sequence. This procedure is adaptable for other types of mutations, and for other phage-bacteria combinations for which appropriate strains and plasmids are available.

Although cells of Bacillus subtilis continue to grow after being infected by bacteriophage SPO1, they do not undergo cell division. The product of SPO1 gene 56 is necessary and sufficient for this inhibition of cell division. GP56 inhibits cell division when expressed in uninfected B. subtilis, without preventing cell growth, DNA synthesis or chromosome segregation, ultimately causing filamentation and loss of viability. During infection, a gene 56 mutation prevents the inhibition of cell division that occurs in wild-type infection. Under the laboratory conditions used, the gene 56 mutation did not affect burst size, latent period, or other components of the host-takeover process.

Bacteriophage genes offer a potential resource for development of new antibiotics. Here, we identify at least six genes of Staphylococcus aureus phage Sb-1 that have bactericidal activity when expressed in Escherichia coli. Since the natural host is gram-positive, and E. coli is gram-negative, it is likely that a variety of quite different bacterial pathogens would be susceptible to each of these bactericidal activities, which therefore might serve as the basis for development of new wide-spectrum antibiotics. We show that two of these gene products target E. coli protein synthesis.

A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.

Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses. Two of these genes, e3 and e22, were cloned and sequenced. E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. Each gene was immediately downstream of a very active early promoter. Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded. An e3 nonsense mutation severely retarded the degradation of e3 RNA. Expression of a plasmid-borne e3 gene, in either B. subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation. However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection. We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities.

Six children of similar ethnic origin with congenital myopathy, cleft palate, malignant hyperthermia (or susceptibility to malignant hyperthermia), and skeletal anomalies are presented. The findings are remarkably consistent among our patients, 3 of whom were related. This syndrome is likely to be inherited as an autosomal recessive trait. Children with this disorder are likely to undergo surgery with general anesthesia for facial or skeletal deformities and should be recognized as predisposed to developing malignant hyperthermia.

We describe a temperature-sensitive mutation of Bacillus subtilis which appears to affect a host function necessary for late protein synthesis during infection by bacteriophage SP82. When cells carrying this mutation are infected by SP82 at the restrictive temperature, synthesis of the late proteins responsible for serum-blocking power and lytic activity is inhibited, while relatively normal synthesis of an early enzyme continues, even at late times.

Normal levels of lytic activity are elicited by SP82 infection of Bacillus subtilis even in the absence of detectable SP82 DNA synthesis. This is contrary to the regulatory patterns of the lysozymes that are known to be phage specific, but is consistent with the hypothesis that the SP82 lytic enzyme is host specific. Normal levels of lytic activity are not, however, sufficient for lysis in SP82 infection. An additional function is required, whose activity can be prevented by an SP82 mutation that blocks DNA synthesis. A host mutation is described which renders this function unnecessary. The strain carrying this mutation can be lysed by SP82 in the absence of detectable DNA synthesis.

Extractable lytic activity in competent cells of Bacillus subtilis 168 was markedly increased after treatment with homologous or heterologous deoxyribonucleic acid (DNA). This increase was prevented by deoxyribonuclease, and did not occur with B. subtilis W23 or with noncompetent B. subtilis 168 cells, neither of which take up DNA. Although the deoxyribonuclease-sensitive step in DNA uptake was completed within 10 min, the increase in lytic activity did not begin until more than 30 min after the addition of DNA. The increase was prevented by any of several antibiotics. These results are discussed in relation to the mechanisms for the uptake of transforming DNA and the lysis of transfected cells.

A nonsense mutation in SPO1 gene 40 prevented normal shutoff of both host DNA and host RNA synthesis, showing that gp40 is required for the normal occurrence of both shutoffs. A gene 39 nonsense mutation caused accelerated shutoff of both host DNA and host RNA synthesis (aided by a gene 38 nonsense mutation), showing that gp39 (aided by gp38) limits the rate at which both shutoffs occur. The 40− mutation suppressed the accelerative effects of the 39− and 38− mutations, showing that gp40 also plays an essential role in the accelerated shutoffs. To the best of our knowledge, proteins with the particular activities implied for gp39 and gp40 have not been identified in any other bacteriophage. SPO1 has at least three different mechanisms that have the effect of delaying the shutoff of host DNA and RNA synthesis.

In mixed infections with Bacillus subtilis phages SP82 and SP01, the SP82 genotype is predominant among the progeny. This predominance is determined by a specific region of the genome, the pos region, which apparently is located near genes 29 to 32 (by the SP01 numbering system). Recombination between SP82 and SP01 yields phage which have both the SP82 pos region and an SP01 mutation. This mutation then behaves in mixed infection as if it were part of an SP82 genome.

This study was performed to evaluate an automatic method of motor unit action potential (MUAP) analysis developed in our laboratory. MUAPs were recorded from the biceps brachii muscle of 68 normal subjects and 122 patients with nerve or muscle disease. The values of mean MUAP durations from normal subjects obtained by automatic analysis were similar to those reported in the literature. However, the normal range of MUAP amplitude and the incidence of polyphasic MUAPs were much higher. Normal ranges of mean MUAP area, area/amplitude ratio, and the number of turns were also defined. Automatic analysis demonstrated an abnormality of at least one MUAP feature in 70% of patients. There was concordance between automated analysis and visual assessment of MUAPs in 76% of patients with neuropathy but in only 50% of patients with myopathy. The relationships between different MUAP features seen in neuropathy and myopathy are explained in physiologic terms.

LONG SHARP foreign bodies in the stomach are alarming to patients, their relatives, and their physicians. Of course, they are potentially dangerous but most of them pass through the gastrointestinal tract without difficulty. When lodged in the esophagus they should be removed, usually by esophagoscopy but occasionally, in difficult cases, by thoracotomy. 1-3 Once they have safely reached the stomach gastroscopic removal is more difficult, normal passage of the foreign body is more likely, and the best policy is, usually, watchful waiting under close hospital and x-ray supervision. Such a policy was successfully carried out in the following case, which is reported because of its general interest and for reassurance of physicians in other similar cases. A 15-year-old boy was admitted to the hospital 5½ hours after swallowing a 6.5 cm. (2.5 in.) corsage pin. He had been asymptomatic since the accident and was asymptomatic on admission. Physical examination