Lawrence J. Shimkets

Geographical distribution and diversity of bacteria associated with natural populations of Drosophila melanogaster.

ABSTRACT Drosophila melanogaster is one of the most widely used model systems in biology. However, little is known about its associated bacterial community. As a first step towards understanding these communities, we compared bacterial 16S rRNA gene sequence libraries recovered from 11 natural populations of adult D. melanogaster. Bacteria from these sequence libraries were grouped into 74 distinct taxa, spanning the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were unevenly spread across host populations. Summed across populations, the distribution of abundance of genera was closely fit by a power law. We observed differences among host population locations both in bacterial community richness and in composition. Despite this significant spatial variation, no relationship was observed between species richness and a variety of abiotic factors, such as temperature and latitude. Overall, bacterial communities associated with adult D. melanogaster hosts are diverse and differ across host populations.

Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility

Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.

The Mosaic Genome of Anaeromyxobacter dehalogenans Strain 2CP-C Suggests an Aerobic Common Ancestor to the Delta-Proteobacteria

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta-Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb 3 oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.

Kineococcus radiotolerans sp. nov., a radiation-resistant, gram-positive bacterium.

A gram-type positive, motile, coccus-shaped organism was isolated from a radioactive work area. Strain SRS30216T is an orange-pigmented bacterium that is catalase-positive, oxidase-negative and urease-negative. The orange pigment is most likely a carotenoid with absorption peaks at approximately 444, 471 and 501 nm. Cells normally grew in clusters, but individual, motile, flagellated cells were also observed. Growth of strain SRS30216T occurred at temperatures between 11 and 41 degrees C, between pH 5 and 9 and at NaCl concentrations up to and including 5%. Fatty acid composition was limited, with >90% of the fatty acids being anteiso 15:0. Alkenes of 19-24 carbons in length were detected during examination of the neutral lipids. Strain SRS30216T demonstrated high levels of resistance to gamma-radiation and desiccation. The most closely related recognized species is Kineococcus aurantiacus RA 333T, which is 93% similar in 16S rDNA sequence. DNA-DNA hybridization revealed only 31% similarity between these two organisms. It is proposed that SRS30216T (= ATCC BAA-149T = DSM 14245T) represents the type strain of a novel species in the genus Kineococcus, Kineococcus radiotolerans sp. nov..

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

SummaryThe myxobacteria are among the simplest organisms with a developmental cycle that is dependent on cell cooperation, and they provide an outstanding system with which to study genes involved in cell interactions. Myxococcus xanthus cells which acquire a csg mutation (formerly known as spoC) lose three different traits, the ability to sporulate, the ability to stimulate adjacent Csg cells to sporulate, and the ability to ripple. The boundaries of the csg locus were determined by transferring a recombinant DNA molecule containing all or part of the locus to Csg mutants and examining the sporulation and rippling phenotypes of the transductants. Three methods were used to integrate the csg locus into the chromosome. First, the entire molecule was integrated into the chromosome by a single homologous crossover. Second, a portion of the molecule was integrated into the chromosome by two flanking homologous crossovers. Third, the entire molecule was integrated into the chromosome by site-specific recombination at a bacteriophage attachment site. Together, these techniques suggested that all of the functions of the csg locus are carried on a DNA fragment of 1.9 kbp or less. The locus appears to contain two smaller units of function. Transposon insertions or deletions in the right end of the locus disrupted sporulation and intercellular complementation of Csg mutants for sporulation, but did not disrupt rippling. The intercellular complementation of Csg mutants may reflect a natural and necessary step in the sporulation of wild-type cells, since the ability to sporulate and the ability to stimulate Csg mutants to sporulate were inseparable by any of these methods.

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.

An Extracellular Matrix-Associated Zinc Metalloprotease Is Required for Dilauroyl Phosphatidylethanolamine Chemotactic Excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.

Methylosarcina fibrata gen. nov., sp. nov. and Methylosarcina quisquiliarum sp. nov., novel type I methanotrophs

Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

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.

Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216.

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energys Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible.