Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca.
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Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part. (+info)
The Mycobacterium tuberculosis extracytoplasmic-function sigma factor SigL regulates polyketide synthases and secreted or membrane proteins and is required for virulence.
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Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the -35 region and CGTgtc in the -10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis. (+info)
Rejection of impassable substrates by Yersinia type III secretion machines.
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Type III machines of pathogenic Yersinia spp. transport Yop proteins across the bacterial envelope into host cells. Translational fusions of yopE to the dihydrofolate reductase gene (dhfr) or the beta-galactosidase gene (lacZ) generate hybrid proteins that block type III injection of Yop proteins into host cells, consistent with the canonical view that impassable DHFR and LacZ hybrids jam secretion machines. Mutations in repressors of posttranscriptional gene regulation, Yersinia enterocolitica yscM1 and yscM2 as well as Yersinia pestis lcrQ, relieve the YopE-DHFR-imposed blockade and restore type III injection into host cells. Genetic suppression of the type III blockade does not, however, promote YopE-DHFR secretion. A model is proposed whereby rejection of YopE-DHFR from the secretion pathway inhibits type III gene expression. (+info)
The influence of anaesthesia on the tensile properties of spider silk.
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In this study of the effect of anaesthesia on both the forced silking process and on the properties of the retrieved silk fibres, a monitored forced silking process enables the silking force to be measured during the whole process. Silk samples were tensile-tested and their diameters measured. Force-displacement curves and stress-strain curves were drawn. The evolution of the silking process of anaesthetized spiders is found to be complex, but it sheds light on the details of the spinning mechanism of spider silk. (+info)
The relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking.
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BACKGROUND: Although rhinovirus (RV) respiratory infections trigger asthma exacerbations, the etiologic association between this virus and severe exacerbations, as well as the clinical characteristics of adults at risk for RV-associated asthma that necessitates hospitalization, have not been established. METHODS: During 1999-2003, we conducted a cohort study of 101 adults prospectively enrolled at hospital admission for an asthma exacerbation. Patient characteristics and frequencies of RV in nasal specimens were analyzed, by reverse-transcription polymerase chain reaction (RT-PCR), at asthma-related hospital admission and at a 3-month convalescent follow-up visit. RESULTS: RV was detected by RT-PCR in 21% of hospitalized patients over a 4-year period and in 1.3% of patients who returned for a 3-month follow-up visit. RV detection was strongly associated with hospitalization for asthma (adjusted odds ratio [OR], 15.1 [95% confidence interval {CI}, 1.88-121.4]). After adjustment for baseline asthma severity, RV-positive patients were more likely than RV-negative patients to be current smokers and nonusers of inhaled corticosteroids (ICSs) (adjusted OR, 11.18 [95% CI, 2.37-52.81]; P=.002). CONCLUSIONS: RV respiratory infection is an etiologic agent in severe asthma exacerbations necessitating hospitalization in adults. Compared with hospitalized patients with asthma who were RV negative, RV-positive patients were significantly more likely to be smokers and nonusers of ICSs. (+info)
JC virus detection in bodily fluids: clues to transmission.
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JC virus in saliva, oropharyngeal fluid, blood, and urine samples obtained from 58 human immunodeficiency virus-infected persons and 58 matched controls was investigated by performing quantitative polymerase chain reaction. JC virus was rarely present in oropharyngeal fluid and blood samples, even in those obtained from immunosuppressed individuals, but it was commonly detected in urine samples from both groups, suggesting that urine contributes to transmission. (+info)
The fission yeast Chs2 protein interacts with the type-II myosin Myo3p and is required for the integrity of the actomyosin ring.
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In Schizosaccharomyces pombe cytokinesis requires the function of a contractile actomyosin ring. Fission yeast Chs2p is a transmembrane protein structurally similar to chitin synthases that lacks such enzymatic activity. Chs2p localisation and assembly into a ring that contracts during division requires the general system for polarised secretion, some components of the actomyosin ring, and an active septation initiation network. Chs2p interacts physically with the type-II myosin Myo3p revealing a physical link between the plasma membrane and the ring. In chs2Delta mutants, actomyosin ring integrity is compromised during the last stages of contraction and it remains longer in the midzone. In synchronous cultures, chs2Delta cells exhibit a delay in septation with respect to the control strain. All these results show that Chs2p participates in the correct functioning of the medial ring. (+info)
A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitor.
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Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single-copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I-II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed. (+info)