Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Pathogenic microorganisms affecting plant health are a major and chronic threat to food production and ecosystem stability worldwide As agricultural production intensified over the past few decades, producers became more and more dependent on agrochemicals as a relatively reliable method of crop

Use of Plant Growth-Promoting Bacteria for Biocontrol of Plant Diseases: Principles, Mechanisms of Action, and Future Prospects

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

Metagenomics: Application of Genomics to Uncultured Microorganisms

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.

PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light

Plants adapt to environmental stresses through specific genetic responses. The molecular mechanisms associated with signal transduction, leading to changes in gene expression early in the stress response, are largely unknown. It is clear, however, that gene expression associated with acclimatory responses is sensitive to the redox state of the cell. Of the many components which contribute to the redox balance of the cell, two factors have been shown to be crucial in mediating stress responses. Thiol/disulphide exchange reactions, particularly involving the glutathione pool and the generation of the oxidant H2O2, are central components of signal transduction in both environmental and biotic stresses. These molecules are multifunctional triggers, modulating metabolism and gene expression. Both are able to cross biological membranes and diffuse or be transported long distances from their sites of origin. Glutathione and H2O2 may act alone or in unison, in intracellular and systemic signalling systems, to achieve acclimation and tolerance to biotic and abiotic stresses.

Hydrogen peroxide‐ and glutathione‐associated mechanisms of acclimatory stress tolerance and signalling

http://www.protocol-online.org/forums/uploads/monthly_05_2010/msg-6517-1275080753.ipb

An improved suicide vector for construction of chromosomal insertion mutations in bacteria.

A hallmark characteristic of species of Aeromonas is their ability to secrete a wide variety of enzymes associated with pathogenicity and environmental adaptability. Among the most intensively studied are beta-lactamases, lipases, hemolytic enterotoxins, proteases, chitinases, nucleases and amylases. Multiple copies of genes encoding each type of enzyme provide additional biological diversity. Except for the chitinases, these multiple copies show little evolutionary relatedness at the DNA level and only limited similarity at the protein level. Indeed a number of the genes, such as nuclease H of A. hydrophila, have no similarity to known prokaryotic or eukaryotic sequences. The challenge is to determine how these genes evolved, where they originated and why Aeromonas possesses them in such abundance and variety.

Secreted enzymes of Aeromonas

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

https://espace.library.uq.edu.au/view/UQ:72277/UQ72277_OA.pdf

Isolation and Characterization of Integron-Containing Bacteria without Antibiotic Selection

A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid. The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1.

Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus.

Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR. The ppsR gene alone is sufficient for photopigment suppression. Inactivation of the R. sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments. The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins. Removal of the helix-turn-helix domain renders PpsR nonfunctional. The promoter of ppsR is located within the coding region of the upstream orf-192 gene. When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli. An R. sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E. coli. This putative promoter activity could be suppressed by PpsR in both E. coli and R. sphaeroides. These results suggest that PpsR is a transcriptional repressor. It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R. sphaeroides and R. capsulatus photosynthesis genes.

https://espace.library.uq.edu.au/view/UQ:389390/UQ389390_OA.pdf

Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides.

John M. Pemberton

Papers

An improved suicide vector for construction of chromosomal insertion mutations in bacteria.

Secreted enzymes of Aeromonas

Isolation and Characterization of Integron-Containing Bacteria without Antibiotic Selection

Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus.

Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides.