Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds. Molecular self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecular surfaces in biological aggregates is a central concern in structural biochemistry. Self-assembly is also emerging as a new strategy in chemical synthesis, with the potential of generating nonbiological structures with dimensions of 1 to 10(2) nanometers (with molecular weights of 10(4) to 10(10) daltons). Structures in the upper part of this range of sizes are presently inaccessible through chemical synthesis, and the ability to prepare them would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.

Molecular self-assembly and nanochemistry: A chemical strategy for the synthesis of nanostructures

The targeting of nucleases to specific DNA sequences facilitates genome editing. Recent work demonstrated that the CRISPR-associated (Cas) nuclease Cas9 can be targeted to sequences in vitro simply by modifying a short7 CRISPR RNA (crRNA) guide. Here we use this CRISPR-Cas system to introduce marker-free mutations in Streptococcus pneumoniae and Escherichia coli. The approach involves re-programming Cas9 by using a crRNA complementary to a target chromosomal locus and introducing a template DNA harboring a desired mutation and an altered crRNA recognition site for recombination with the target locus. We exhaustively analyze Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements. Alone or together with recombineering, CRISPR assisted editing induces recombination at the targeted locus and kills non-edited cells leading to a recovery of close to a 100% of edited cells. Multiple crRNA can be used to modify several loci simultaneously. Our results show that CRISPR-mediated genome editing only requires programming of the crRNA and template sequences and thus constitutes a useful tool for genetic engineering.

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RNA-guided editing of bacterial genomes using CRISPR-Cas systems

Communities of microscopic plant life, or phytoplankton, dominate the Earth's aquatic ecosystems. This important new book by Colin Reynolds covers the adaptations, physiology and population dynamics of phytoplankton communities in lakes and rivers and oceans. It provides basic information on composition, morphology and physiology of the main phyletic groups represented in marine and freshwater systems and in addition reviews recent advances in community ecology, developing an appreciation of assembly processes, co-existence and competition, disturbance and diversity. Although focussed on one group of organisms, the book develops many concepts relevant to ecology in the broadest sense, and as such will appeal to graduate students and researchers in ecology, limnology and oceanography.

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The Ecology of Phytoplankton

Diverse bacterial species possess the ability to produce the auxin phytohormone indole-3-acetic acid (IAA). Different biosynthesis pathways have been identified and redundancy for IAA biosynthesis is widespread among plant-associated bacteria. Interactions between IAA-producing bacteria and plants lead to diverse outcomes on the plant side, varying from pathogenesis to phytostimulation. Reviewing the role of bacterial IAA in different microorganism–plant interactions highlights the fact that bacteria use this phytohormone to interact with plants as part of their colonization strategy, including phytostimulation and circumvention of basal plant defense mechanisms. Moreover, several recent reports indicate that IAA can also be a signaling molecule in bacteria and therefore can have a direct effect on bacterial physiology. This review discusses past and recent data, and emerging views on IAA, a well-known phytohormone, as a microbial metabolic and signaling molecule.

Indole-3-acetic acid in microbial and microorganism-plant signaling.

The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2005.04587.x

Bacillus subtilis antibiotics: structures, syntheses and specific functions

Structural Basis for Transcription Regulation by Alarmone ppGpp

Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2006.05585.x

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria.

The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.

Polyketide Synthase Gene Coupled to the Peptide Synthetase Module Involved in the Biosynthesis of the Cyclic Heptapeptide Microcystin

Genome sequencing of Streptomyces, myxobacteria, and fungi showed that although each strain contains genes that encode the enzymes to synthesize a plethora of potential secondary metabolites, only a fraction are expressed during fermentation. Interest has therefore grown in the activation of these cryptic pathways. We review current progress on this topic, describing concepts for activating silent genes, utilization of “natural” mutant-type RNA polymerases and rare earth elements, and the applicability of ribosome engineering to myxobacteria and fungi, the microbial groups known as excellent searching sources, as well as actinomycetes, for secondary metabolites.

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New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

Kozo Ochi

Papers

Structural Basis for Transcription Regulation by Alarmone ppGpp

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria.

Polyketide Synthase Gene Coupled to the Peptide Synthetase Module Involved in the Biosynthesis of the Cyclic Heptapeptide Microcystin

New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).