Induction of beta-lactamase influences the course of development in Myxococcus xanthus.
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Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus. (+info)
Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2).
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The enhanced green fluorescent protein (EGFP) gene is a modified version of the green fluorescent protein gene of the jellyfish Aequorea victoria with a codon usage that corresponds well to that found in many GC-rich streptomycete genes. Here the use of EGFP as a reporter for the analysis of spatially and temporally regulated gene expression in Streptomyces coelicolor A3(2) is demonstrated. The EGFP gene was inserted into plasmids that can replicate in Escherichia coli, greatly facilitating the construction of EGFP gene fusions. The plasmids can be transferred readily to S. coelicolor by conjugation, whereupon two of them (pIJ8630 and pIJ8660) integrate at the chromosomal attachment site for the temperate phage phiC31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a sigma factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by S. coelicolor. While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redD occurred only in ageing substrate mycelium. A further plasmid derivative (pIJ8668) was made that lacks the phiC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations. (+info)
Association of early sporulation genes with suggested developmental decision points in Streptomyces coelicolor A3(2).
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Cytological analysis of a series of Streptomyces coelicolor A3(2) mutants with disruptions of early sporulation (whi, for white aerial mycelium) genes in an isogenic background has provided new information about the role of whiH, and confirmed and extended previous knowledge about whiG, whiA and whiB. The characteristic straight aerial hyphae of whiG mutants contained normally spaced vegetative-like septa, while mutants in whiA or whiB had abnormally long and coiled aerial hyphae almost devoid of septation. whiG, whiA and whiB were all absolutely required for sporulation septation, and for all visible signs of nucleoid condensation and partitioning and other changes associated with later stages of sporulation. On the other hand, whiH appeared to enhance low basal levels of these processes. Thus, whiH mutant aerial hyphae were divided into loosely coiled fragments of variable sizes by what appeared to be a few sporulation septa. These fragments showed some spore-like characteristics and contained condensed and aberrantly partitioned nucleoids. whiG, whiA and whiB were epistatic to whiH on the criterion that they prevented such fragments from forming in double mutants. These spore-like features and the synthesis of clearly detectable levels of the whiE-directed grey spore pigment were not due to any residual activity of previously studied whiH alleles since they were retained by a constructed whiH null mutant. A model is presented that explains the mutant phenotypes by proposing two early developmental decision points involved in commitment to sporulation septation, one requiring whiG and the other requiring whiA and whiB. (+info)
Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.
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In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli. (+info)
Experimental studies on environmental contamination with infected blood during haemodialysis.
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To assess the relative importance of different postulated modes of spread of hepatitis B in dialysis units, blood charged with various tracer organisms was used in simulated haemodialysis runs in four laboratories, and the resulting contamination of equipment and environment was measured semi-quantitatively. Some airborne spread of the tracer organism occurred when tubing containing contaminated blood was needled as the "patient" went on and came off the dialyser. Virtually no small airborne particles could be demonstrated however in simulated emergencies in which a blood line was disconnected, or even when bottles of blood were dropped on to a hard floor from a height of 2 metres. Bacillus globigii spores from contaminated blood leaked in small numbers into the dialysing fluid through apparently intact coils. T3 phage, with a particle size of the same order as hepatitis B virus, passed in small quantities through the membrane of a Kiil dialyser from blood to dialysing fluid and also in the reverse direction when added to the header tank. A number of other dialysers were also permeable to phage. Visual assessment of the appropriate moment for inserting the venous line into the "patient" at the onset of dialysis was shown to be unreliable, as the displaced fluid from the end of the venous line was already contaminated before it contained visible red blood cells. Considerable contamination of exposed surfaces and of the buttons on the proportionating unit cabinet occurred. Minor visible splashing of blood was a common-place of the laboratory experiments and was shown to be also a common event during routine haemodialysis in two of the dialysis units taking part in the studies. (+info)
Streptomyces malaysiensis sp. nov., a new streptomycete species with rugose, ornamented spores.
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The taxonomic position of a streptomycete strain isolated from Malaysian soil was established using a polyphasic approach. The organism, designated strain ATB-11T, was found to have chemical and morphological properties consistent with its classification in the genus Streptomyces. An almost complete 16S rRNA gene (rDNA) sequence determined for the test strain was compared with those of previously studied streptomycetes by using two treeing algorithms. The 16S rDNA sequence data not only supported classification of the strain in the genus Streptomyces but also showed that it formed a distinct phyletic line. At maturity, the aerial hyphae of strain ATB-11T differentiated into tight, spiral chains of rugose, cylindrical spores. The organism was readily distinguished from representatives of validly described Streptomyces species with rugose spores by using a combination of phenotypic features. It is proposed, therefore, that strain ATB-11T be classified in the genus Streptomyces as Streptomyces malaysiensis sp. nov. (+info)
A novel surfactant nanoemulsion with broad-spectrum sporicidal activity against Bacillus species.
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Two nontoxic, antimicrobial nanoemulsions, BCTP and BCTP 401, have been developed. These emulsions are composed of detergents and oils in 80% water. BCTP diluted up to 1:1000 inactivated>90% of Bacillus anthracis spores in 4 h and was also sporicidal against three other Bacillus species. This sporicidal activity is due to disruption of the spore coat after initiation of germination without complete outgrowth. BCTP 401 diluted 1:1000 had greater activity than BCTP against Bacillus spores and had an onset of action of <30 min. Mixing BCTP or BCTP 401 with Bacillus cereus prior to subcutaneous injection in mice reduced the resulting skin lesion by 99%. Wound irrigation with BCTP 1 h after spore inoculation yielded a 98% reduction in skin lesion size, and mortality was reduced 3-fold. These nanoemulsion formulas are stable, easily dispersed, nonirritant, and nontoxic compared with other available sporicidal agents. (+info)
Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE.
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The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat's outer layer. We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly. (+info)