Robust Stoichiometry of FliW-CsrA Governs Flagellin Homeostasis and Cytoplasmic Organization in Bacillus subtilis.

Flagellin (Hag) is one of the most abundant proteins in Bacillus subtilis Here we show that each flagellar filament is assembled from ∼12,000 Hag monomers and that there is a cytoplasmic pool of Hag that is restricted to 5% of the total. Hag is thought to be restricted at the level of translation by a partner-switching mechanism involving FliW and the homodimeric RNA-binding protein CsrA (CsrAdimer). We further show that the mechanism of translation inhibition is hypersensitive due to a 1:1 ratio of Hag to FliW, a 1:1 inhibitory ratio of FliW to CsrAdimer, and a nearly 1:1 ratio of CsrAdimer to hag transcripts. Equimolarity of all components couples single-molecule detection of Hag export to compensatory translation and causes cytoplasmic Hag concentrations to oscillate around the level of FliW. We found that stoichiometry is ensured by genetic architecture, translational coupling, and the ability of CsrAdimer to restrict hag transcript accumulation. We further show that homeostasis prevents Hag hyperaccumulation that would otherwise cause severe defects in intracellular architecture, perhaps due to increased molecular crowding. We note that FliW-CsrA-mediated structural homeostasis has similarities to that seen with some toxin-antitoxin systems.IMPORTANCE The intracellular concentration of flagellar filament protein Hag is restricted by the Hag-FliW-CsrA system in B. subtilis Here we show that the Hag-FliW-CsrAdimer system functions at nearly 1:1:1 stoichiometry and that the system is both robust with respect to perturbation and hypersensitive to the Hag intracellular concentration. Moreover, restriction of cytoplasmic Hag levels is important for maintaining proper intracellular architecture, as artificial Hag hyperaccumulation led to generalized spatial defects and a high frequency of minicell production. The Hag-FliW-CsrA system is conserved in the deeper branches of bacterial phylogeny, and we note that the Hag-FliW-CsrA "homeostasis module" resembles a toxin-antitoxin system where, by analogy, CsrA is the "toxin," FliW is the "antitoxin," and Hag is the target.

ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis.

The ancestral Bacillus subtilis strain 3610 contains an 84-kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1 or 2 copies per cell, increases in copy number nearly 100-fold when cells are treated with the DNA-damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32-borne prophage genes. ZpdN, a sigma factor homolog encoded by pBS32, was required for the plasmid response to DNA damage, and artificial expression of ZpdN was sufficient to induce pBS32 hyperreplication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH, suggesting that the plasmid was packaged into a phagelike particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killing B. subtilis cells extracellularly. We hypothesize that pBS32-mediated cell death releases a phagelike particle that is defective and unstable. IMPORTANCE: Prophages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 of Bacillus subtilis encodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

Identification of a developmental chemoattractant in Myxococcus xanthus through metabolic engineering.

Fruiting body formation of Myxococcus xanthus requires the ordered migration of tens of thousands of cells by using a form of surface motility known as gliding and chemical signal(s) that have yet to be elucidated. Directed movement is regulated by phosphatidylethanolamine (PE) purified from M. xanthus cell membranes. Because the purified PE preparation contains a remarkably diverse mixture of fatty acids, metabolic engineering was used to elucidate the biologically active fatty acid component. The mutational block in an esg mutant, which renders it defective in producing primers for branched-chain fatty acid biosynthesis, was bypassed with one of a series of primers that enriches for a particular family of branched-chain fatty acids. Each PE enrichment was observed for chemotactic activity by using an excitation assay and for fatty acid content. The excitation activity of a PE preparation was generally proportional with the concentration of the fatty acid 16:1 omega 5c. 1,2-O-Bis[11-(Z)-hexadecenoyl]-sn-glycero-3-phosphoethanolamine (PE-16:1 omega 5c/16:1 omega 5c) was synthesized and elicited an excitation peak at 2 ng. This peak activity occurred at a 1,000-fold lower concentration than dilauroyl PE (PE-12:0/12:0) and the peak magnitude was 2-fold higher. PE containing 16:1 omega 5c is likely to play a role in development because it is active at physiological concentrations and only under developmental conditions.

Lipid chemotaxis and signal transduction in Myxococcus xanthus.

The lipid phosphatidylethanolamine (PE) is the first chemoattractant to be described for a surface-motile bacterium. In Myxococcus xanthus, the specific activity of PE is determined by its fatty acid components. Two active species have been identified: dilauroyl PE and dioleoyl PE. Excitation to dilauroyl PE requires fibril appendages and the presence of two cytoplasmic chemotaxis systems, of which one (Dif) appears to mediate excitation and the other (Frz) appears to mediate adaptation. A possible mechanism for fibril-mediated signal transduction is discussed, along with the potential roles for PE chemotaxis in the context of the M. xanthus life cycle.

Pseudomonas aeruginosa exhibits directed twitching motility up phosphatidylethanolamine gradients.

Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type P. aeruginosa, but not a pilJ pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing P. aeruginosa motility while many other species were inactive. P. aeruginosa PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.

Myxococcus xanthus fibril appendages are essential for excitation by a phospholipid attractant.

Isolated (A-motile) Myxococcus xanthus cells glide over solid surfaces and display excitation, a suppression of direction reversals, when presented with phosphatidylethanolamine (PE) purified from its own membranes or synthetic dilauroyl PE and dioleoyl PE. Although the mechanism of PE signal transduction is unknown, we hypothesized that M. xanthus might use surface-associated factors to detect exogenous PE to prevent endogenous lipids from self-stimulating the sensory system. Peritrichous protein and polysaccharide appendages called fibrils were correlated with dilauroyl PE excitation. Wild-type cells treated with Congo red, an inhibitor of fibril assembly, and mutants defective in fibril biosynthesis showed an elevated reversal period, which suggested that fibrils regulate the gliding motor. Furthermore, the loss of fibrils resulted in loss of excitation to dilauroyl PE but not dioleoyl PE. Restoration of fibril production to these mutants restored the dilauroyl PE response. In addition, the dif cytoplasmic signal transduction system and starvation conditions were required for dilauroyl PE excitation. The chemically specific nature of the response and the dependence on the dif system suggests that fibrils define a novel sensory organelle whose evolution may have been necessary to prevent autostimulation by endogenous membrane lipids. Because the hydrophobic nature of dilauroyl PE would be inaccessible to periplasmic chemosensors, we suggest that fibrils act as extracellular signal transducers to probe surfaces for insoluble chemical signals.

Clinical update on 10 children treated with intralesional cidofovir injections for severe recurrent respiratory papillomatosis.

OBJECTIVES: To continue assessment of the benefits and risks of intralesional administration of cidofovir, an acyclic nucleoside phosphonate, for treating severe recurrent respiratory papillomatosis (RRP) in pediatric patients, and to discuss guidelines for larger prospective multi-institutional studies of the use of cidofovir. DESIGN: Prospective case series. SETTING: Tertiary care children's hospital. PATIENTS: A total of 10 patients with severe RRP (defined as requiring debulking procedures to maintain airway patency at least once a month) underwent intralesional cidofovir therapy. The original 5 patients have received more than 1 year of follow-up since their last cidofovir injection, and 5 subsequent patients have been treated with a revised injection protocol. INTERVENTION: Microsuspension laryngoscopy with intralesional injection of cidofovir after repetitive carbon dioxide laser treatments and mechanical debulking of papillomas. MAIN OUTCOME MEASURES: Papilloma stage at the time of serial laryngoscopies. Histologic examination of biopsy specimens of laryngeal tissue obtained 1 year or more after last cidofovir injection. RESULTS: There was evidence of marked improvement in the 4 of the 5 new patients enrolled under the revised injection protocol, continuation of a disease-free state in 1 of the original 5 patients, and sustained improvement in 4 of the 5 original patients, resulting in a significantly reduced interval of intervention. CONCLUSIONS: Intralesional cidofovir therapy continues to show benefit in the treatment of severe RRP in pediatric patients. Safety profiles have not been fully established, but current histologic data are reassuring.

An atypical presentation of Kawasaki disease resembling a retropharyngeal abscess.

Kawasaki disease is an acute systemic inflammatory disease, which occurs in children less than 10 years of age. The etiology of the disorder is unknown. Diagnosis is based upon clinician's recognition of a symptom pattern that includes high fevers, oral cavity changes, polymorphous rash, conjunctival injection, and cervical adenopathy. Most feared are the cardiac manifestations of Kawasaki syndrome, which result in an overall mortality rate of 2% in patients. Because of the common presenting symptoms, the otolaryngologist often plays a central role in early diagnosis of the disorder. The following case report describes a patient with Kawasaki disease whose initial presentation mimicked a retropharyngeal abscess. A literature review details the common and atypical early signs and symptoms of Kawasaki disease.

Intralesional cidofovir for recurrent respiratory papillomatosis in children.

OBJECTIVE: To assess the potential benefit of intralesional administration of cidofovir, an acyclic nucleoside phosphonate with activity against several DNA viruses, for treating severe respiratory papillomas in pediatric patients. DESIGN: Prospective case series. SETTING: Tertiary care children's hospitals. PATIENTS: Five pediatric patients with severe recurrent respiratory papillomatosis requiring laryngoscopy with carbon dioxide laser therapy more frequently than once a month to maintain airway patency. Each patient underwent between 12 and 33 laryngoscopies with laser treatment prior to being injected with cidofovir. INTERVENTION: Microsuspension laryngoscopy with intralesional injection of cidofovir (Vistide) in conjunction with mechanical debulking and carbon dioxide laser of papillomas. MAIN OUTCOME MEASURE: Papilloma stage at time of serial laryngoscopies. RESULTS: One patient was disease free and 3 patients demonstrated a dramatic response to adjuvant therapy with cidofovir at the 9-month follow-up visit after the last injection of cidofovir. One patient showed an improvement in papilloma stage that was possibly related to concurrent therapy with interferon. CONCLUSIONS: Intralesional injection of cidofovir seems to be of benefit in the treatment of severe respiratory papillomatosis in pediatric patients. Larger prospective studies with longer follow-up will be required before cidofovir can be considered an accepted means of managing this difficult disease.

Giant juvenile xanthogranuloma of the tongue.

Juvenile xanthogranulomas (JXGs) are rare, benign, fibrohistiocytic lesions. They usually appear as one or more cutaneous papules on the head, neck, or trunk in infants. Twelve cases of oral JXGs have been reported, four of which involved the tongue. We present a 6-year-old girl with a large tongue mass diagnosed as JXG after an excisional biopsy. Histological and immunohistochemical staining results are presented. This is the first reported case of a giant oral JXG. A review of the literature on these unusual lesions is presented, along with discussion of their differential diagnosis and key aspects of the patient's evaluation, management, and pathological diagnosis.

Evaluation and management of benign, non-congenital tongue masses in children.

Lingual tumors are rare, primarily benign, lesions in the pediatric population. Congenital lesions, such as hemangiomas, lymphatic malformations, dermoids, hamartomas and thyroglossal ducts cysts, are seen more commonly. Primary, non-congenital lingual neoplasms are less common in children. We present three patients with benign lingual neoplasms. Evaluation, management, pathology and follow-up are discussed.

Adenotonsillectomy in the very young patient: cost analysis of two methods of postoperative care.

Postoperative management of the patient younger than 36 months undergoing adenotonsillectomy has been the subject of many debates. Concerns for early postoperative complications such as airway obstruction, emesis, dehydration, and hemorrhage have led many physicians to consider overnight hospitalization following adenotonsillectomy in very young children. Trends in health care management have had increasing focus on cost effective means of treating patients to limit unnecessary expenditure on the part of the patient, physician, and hospital facility. The purpose of this retrospective review was to analyze two methods of early postoperative management in children less than 36 months old undergoing adenotonsillectomy at the Children's Hospital, San Diego from 1992 to 1997. Three hundred and seven cases were reviewed. Same-day discharge was compared with overnight inpatient observation based on the cost analysis of these two methods of postoperative care. Postoperative care was based on length of stay in the recovery room and as an inpatient. Expense of postoperative care was based on cost calculation for the recovery room and overnight hospitalization. Of the 307 patients, 194 went home the day of surgery and 113 were observed overnight in the hospital. Average hospital cost was higher in the outpatient group than in the inpatient group (P < 0.001). This difference reflects longer recovery room stay (350 min) in the outpatient group compared to the inpatient group (108 min) (P < 0.001). Outpatient adenotonsillectomy in the patient under 36 months may be safe; however, prolonged recovery room stays may actually make outpatient surgery less cost-effective than overnight admission. Recovery room costs are significantly higher per unit time than costs of inpatient hospitalization. Further investigation of cost-effective outpatient observation units may improve cost containment in the outpatient surgical setting.

Acute coalescent mastoiditis in an infant: an emerging trend?

There is some evidence to suggest that the incidence and complications of ACM may be increasing. However, in the current era of widespread access to health care and broad-spectrum antibiotics, an intratemporal or intracranial complication from acute otitis media may not initially be suspected. The reported case is significant in that the patient was very young, had no underlying disease or immunocompromise, and did not have a known antecedent acute otitis media. With the emergence of resistant streptococcal species and prolonged survival in immunocompromised patients, the relative incidence of complications caused by acute otitis media will probably continue to rise, making it imperative that we raise our index of suspicion for previously rare infectious complications of relatively common diseases.

Chemotaxis in a gliding bacterium.

Myxococcus xanthus cells exhibit directed motility up phosphatidylethanolamine (PE) gradients, and we suggest that PE behaves as a chemoattractant. Computer-assisted stop-motion digital microscopy was used to record cell movements in slide culture. PE decreased cellular reversal frequency with molecular specificity that was correlated with the fatty acid composition. Synthetic dilauroyl (di C12:0) PE and dioleoyl (di C18:1 omega9c) PE suppressed direction reversals and stimulated movement up the gradient. Sensory adaptation occurred about 1 hr after the onset of stimulation. Null mutants in a methylated chemotaxis protein homolog (FrzCD) and a CheA/CheY homolog (FrzE) moved up a PE gradient at a reduced rate. The mutants displayed normal excitation but were defective in adaptation. A dominant, hyper-reversal mutant in the M. xanthus methyl accepting chemotaxis protein homolog, frzCD224, failed to respond to PE stimulation, which argued that PE was a transduced stimulus. Neither dilauroyl PE nor dioleoyl PE is present at high enough concentrations in vegetative or developmental PE to account for all of the chemotactic activity. It appears then that there are additional, as yet unknown, PE species that serve as autoattractants. We report on a discrete phospholipid chemoattractant in a gliding bacterium

Discoordinate pharyngolaryngomalacia.

In cases of severe laryngomalacia, laser division of the aryepiglottic folds (AEFs) or endoscopic supraglottoplasty may be an ineffective solution. Failure of this technique is rare and the reasons for failure are not well established. The purpose of this study was to describe those cases of laryngomalacia in which endoscopic treatment did not reverse the clinical situation. We introduce the concept of discoordinate pharyngolaryngomalacia (DPLM). DPLM was defined as severe laryngomalacia with complete supraglottic collapse during inspiration, without shortened AEFs or redundant mucosa, and with associated pharyngomalacia. Twenty-seven of 82 children with severe laryngomalacia presented a DPLM. Endoscopic treatment was performed in 16 children and the surgical procedure was inadequate to reverse the clinical problem in these patients. In 10 children correction of additional sites of obstruction was required (uvulopharyngopalatoplasty, surgery of choanal atresia, aortopexy). Tracheostomy was necessary in 13 children. Bi-level positive airway pressure (BiPAP) was used successfully in 2 children and tracheotomy was avoided. Treatment still needs to be better defined.

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis.

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)-1(min)-1. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (Ks) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a Ks of 0.20 mM, but the Ks of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport.