Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.     

Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units

The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.     

Chitin utilization by marine bacteria. Chemotaxis to chitin oligosaccharides by Vibrio furnissii.

The adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii (Yu, C., Lee, A., Bassler, B. L., and Roseman, S. (1991) J. Biol. Chem. 266, 24260-24267) probably catalyzes the first step in colonizing chitin. Evidence is presented here for a second step, chemotaxis to chitin hydrolysis products. V. furnissii swarms toward chitin oligomers (GlcNAc)n, n = 1-6, at initial concentrations as low as 10 microM. A modified capillary assay was used for quantitation; the cells exhibit low level constitutive taxis to GlcNAc but not to the oligosaccharides. A mutant defective in the GlcNAc receptor (IINag of the phosphotransferase system) showed inducible taxis to the oligosaccharides. Two (or more) independently inducible receptors with overlapping specificities recognize (GlcNAc)n, n = 2-4. (GlcNAc)5 and (GlcNAc)6 were inactive in the capillary assay; expression of this receptor(s) apparently require special induction conditions. The (GlcNAc)n, n = 1-4, chemoreceptors of V. furnissii may be the most potent reported for bacteria. L-Amino acids were weak, constitutive attractants; glutamine, not known to be an attractant in other bacteria, was the most effective amino acid. The most potent receptor in Escherichia coli, Tar (aspartate), is not expressed in V. furnissii. The chemotactic responses were greatly affected by growth and induction conditions and the presence of nutrients in the assay media. Taxis to GlcNAc and GlcNAc oligomers was optimally induced by growth in lactate medium containing 0.6 mM sugar, while growth on the sugar per se resulted in poor taxis. Chemotaxis to the sugars increased 2- to 3-fold when the cells were starved. Nutrients in the assay medium, especially compounds that feed into or are part of the Krebs cycle, were potent inhibitors of taxis to the sugars and Gln. With the exception of isocitrate, inhibition of taxis correlated with the rate of oxidation of these compounds. The results suggest a link between catabolism and taxis in this organism, i.e. interactions or "cross-talk" between systems that are regulated by protein phosphorylation (Stock, J. A., Ninfa, A. J., and Stock, A. M. (1989) Microbiol. Rev. 53, 450-490).     

Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex

A novel form of mitochondrial DNA (mtDNA) inheritance has previously been documented for the blue mussel (Mytilus edulis). Female mussels inherit their mtDNA solely from their mother while males inherit mtDNA from both their mother and their father. In males, the paternal mtDNA is preferentially amplified so that the male gonad is highly enriched for the paternal mtDNA that is then transmitted from fathers to sons. We demonstrate that this mode of mtDNA inheritance also operates in the closely related species M. galloprovincialis and M. trossulus. The evolutionary relationship between the male and female mtDNA lineages is estimated by phylogenetic analysis of 455 nucleotides from the large subunit ribosomal RNA gene. We have found that the male and female lineages are highly divergent; the divergence of these lineages began prior to the speciation of the three species of blue mussels. Further, the separation between the male and female lineages is estimated to have occurred between 5.3 and 5.7 MYA.      

A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi

Two independent quorum-sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi. Each system is composed of an autoinducer (AI-1 or AI-2) and its cognate sensor (LuxN or LuxQ). The sensors are two-component hybrid kinases, containing both sensor kinase domains and response regulator domains. Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO. LuxO is a member of the response regulator class of the two-component family of signal transduction proteins, and LuxO acts negatively to control luminescence. In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V. harveyi chromosome at the luxO locus. Phenotypical analyses of the resulting mutant V. harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive. Analysis of the lux phenotypes of V. harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO. They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers. Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.     

Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence

Background

The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy.

Methodology/Principal Findings

The marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp) strain increased the growth rate of wild type Vh without significantly altering Vh''s bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants.

Conclusions/Significance

The effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate.