Jon R. Saunders

Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms

A bstractThe application of molecular biological methods to study the diversity and ecology of microorganisms in natural environments has been practiced since the mid-1980s. Since that time many new insights into the composition of uncultivated microbial communities have been gained. Whole groups of organisms that are only known from molecular sequences are now believed to be quantitatively significant in many environments. Molecular methods have also allowed characterization of many long-recognized but poorly understood organisms. These organisms have eluded laboratory cultivation and, hence, have remained enigmatic. This review provides an outline of the main methods used in molecular microbial ecology, and their limitations. Some discoveries, made through the application of molecular biological methods, are highlighted, with reference to morphologically distinctive, uncultivated bacteria; an important biotechnological process (wastewater treatment); and symbiotic relationships between Bacteria, Archaea and Eukarya.

Group Search Optimizer: An Optimization Algorithm Inspired by Animal Searching Behavior

Nature-inspired optimization algorithms, notably evolutionary algorithms (EAs), have been widely used to solve various scientific and engineering problems because of to their simplicity and flexibility. Here we report a novel optimization algorithm, group search optimizer (GSO), which is inspired by animal behavior, especially animal searching behavior. The framework is mainly based on the producer-scrounger model, which assumes that group members search either for ldquofindingrdquo (producer) or for ldquojoiningrdquo (scrounger) opportunities. Based on this framework, concepts from animal searching behavior, e.g., animal scanning mechanisms, are employed metaphorically to design optimum searching strategies for solving continuous optimization problems. When tested against benchmark functions, in low and high dimensions, the GSO algorithm has competitive performance to other EAs in terms of accuracy and convergence speed, especially on high-dimensional multimodal problems. The GSO algorithm is also applied to train artificial neural networks. The promising results on three real-world benchmark problems show the applicability of GSO for problem solving.

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N‐linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O‐linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1‐4‐linked digalactose moiety covalently linked to a 2,4‐diacetamido‐2,4,6‐trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper‐adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.

Pilus‐facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non‐piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper‐adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS‐PAGE. Hyper‐adherent pilins shared unique changes in sequence including substitution of Asn‐113 for Asp‐113 and changes from Asn‐Asp‐Thr‐Asp to Thr‐Asp‐Ala‐Lys at residues 127‐130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N‐glycosylation motif Asn‐X‐Ser/Thr and could potentially be glycosylated post‐translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS‐PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn‐60‐Asn‐61‐Thr‐62 in an adherent variant was replaced with Asp‐60‐Asn‐61‐Ser‐62 in a non‐adherent variant. Fully adherent backswitchers obtained from the non‐adherent variant always regained Asn‐60 but retained Ser‐62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N‐linked glycosylation sites on the pilin subunit.

Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus‐deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep‐2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili‐expressing strains. In addition to these inter‐class functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep‐2 cells (‘non‐adherent’ phenotype; adherence of < 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non‐adherent Pil‐ and Pil‐ bacteria by selection on Chang cells (adherence of 10–25 bacteria per cell). In contrast to epithelial cells, al) variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (<50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus‐facilitated interactions of N. meningitidis with endothelial and epithelial cells.

Bacterial Foraging Algorithm For Dynamic Environments

Optimization in dynamic environments has received great attention in recent years [1]. Different from static optimization problems, its convergence and searching ability is cautiously desired. Over the last two decades, evolutionary algorithms (EAs), designed to solve the static optimization problems, have been comprehensively and intensively investigated. In recent years, as the emergence of another member of the EA family -bacterial foraging algorithm (BFA), the self-adaptability of individuals in the group searching activities has attracted a great deal of interests. In this paper, a BFA aiming for optimization in dynamic environments, called DBFA, is studied. A test bed proposed previously in [2] is adopted to evaluate the performance of DBFA. The simulation studies offer a range of changes in a dynamic environment. The simulation results show that DBFA can adapt to various environmental changes which occur in different probabilities, with both satisfactory accuracy and stability, in comparison with a recent work on bacterial foraging [3].

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

ABSTRACT A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number ofShigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens.

ABSTRACT The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

Plasmid Transfer between the Bacillus thuringiensis Subspecies kurstaki and tenebrionis in Laboratory Culture and Soil and in Lepidopteran and Coleopteran Larvae

ABSTRACT Plasmid transfer between Bacillus thuringiensis subsp.kurstaki HD1 and B. thuringiensis subsp.tenebrionis donor strains and a streptomycin-resistantB. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10−1 transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When aB. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp.tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.