Search

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO 2 . Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Polar Regions are unique and highly prolific ecosystems characterized by extreme environmental gradients. Photosynthetic autotrophs, the base of the food web, have had to adapt physiological mechanisms to maintain growth, reproduction and metabolic activity despite environmental conditions that would shut-down cellular processes in most organisms. High latitudes are characterized by temperatures below the freezing point, complete darkness in winter and continuous light and high UV in the summer. Additionally, sea-ice, an ecological niche exploited by microbes during the long winter seasons when the ocean and land freezes over, is characterized by large salinity fluctuations, limited gas exchange, and highly oxic conditions. The last decade has been an exciting period of insights into the molecular mechanisms behind adaptation of microalgae to the cryosphere facilitated by the advancement of new scientific tools, particularly "omics" techniques. We review recent insights derived from genomics, transcriptomics, and proteomics studies. Genes, proteins and pathways identified from these highly adaptable polar microbes have far-reaching biotechnological applications. Furthermore, they may provide insights into life outside this planet, as well as glimpses into the past. High latitude regions also have disproportionately large inputs into global biogeochemical cycles and are the region most sensitive to climate change.

Dimethylsulfoniopropionate (DMSP) plays important roles in oceanic carbon and sulfur cycling and may significantly impact climate. It is a biomolecule synthesized from the methionine (Met) pathway and proposed to serve various physiological functions to aid in environmental stress adaptation through its compatible solute, cryoprotectant, and antioxidant properties. Yet, the enzymes and mechanisms regulating DMSP production are poorly understood. This study utilized a proteomics approach to investigate protein changes associated with salinity-induced DMSP increases in the model sea-ice diatom Fragilariopsis cylindrus (CCMP 1102). We hypothesized proteins associated with the Met-DMSP biosynthesis pathway would increase in relative abundance when challenged with elevated salinity. To test this hypothesis axenic log-phase cultures initially grown at a salinity of 35 were gradually shifted to a final salinity of 70 over a 24-h period. Intracellular DMSP was measured and two-dimensional gel electrophoresis was used to identify protein changes at 48 h after the shift. Intracellular DMSP increased by approximately 85% in the hypersaline cultures. One-third of the proteins increased under high salinity were associated with amino acid pathways. Three protein isoforms of S-adenosylhomo-cysteine hydrolase, which synthesizes a Met precursor, increased 1.8- to 2.1-fold, two isoforms of S-adenosyl Met synthetase increased 1.9- to 2.5-fold, and S-adenosyl Met methyltransferase increased by 2.8-fold, suggesting active methyl cycle proteins are recruited in the synthesis of DMSP. Proteins from the four enzyme classes of the proposed algal Met transaminase DMSP pathway were among the elevated proteins, supporting our hypothesis and providing candidate genes for future characterization studies.

Strains of Staphylococcus aureus that are resistant to multiple antimicrobial compounds, including most available classes of antibiotics and some antiseptics, are a major threat to patient care owing to their stubborn intransigence to chemotherapy and disinfection. This reality has stimulated extensive efforts to understand the genetic nature of the determinants encoding antimicrobial resistance, together with the mechanisms by which these determinants evolve over time and are spread within bacterial populations. Such studies have benefited from the application of molecular genetics and in recent years, the sequencing of over a dozen complete staphylococcal genomes. It is now evident that the evolution of multiresistance is driven by the acquisition of discrete preformed antimicrobial resistance genes that are exchanged between organisms via horizontal gene transfer. Nonetheless, chromosomal mutation is the catalyst of novel resistance determinants and is likely to have an enhanced influence with the ongoing introduction of synthetic antibiotics.

Interactions between Gossypium spp. and the bacterial pathogen Xanthomonas campestris pv. malvacearum are understood in the context of the gene-for-gene concept. Reviewed here are the genetic basis for cotton resistance, with reference to resistance genes, resistance gene analogs, and bacterial avirulence genes, together with the physiological mechanisms involved in the hypersensitive response to the pathogen, including production of signaling hormones, synthesis of antimicrobial molecules and alteration of host cell structures. This host-pathogen interaction represents the most complex resistance gene/avr gene system yet known and is one of the few in which phytoalexins are known to be specifically localized in HR cells at anti-microbial concentrations.

Verticillium wilt is a vascular disease of cotton (Gossypium spp.) caused by the fungal pathogen Verticillium dahliae. To begin to understand the molecular mechanisms of the disease response in cotton cultivars that display superior wilt tolerance, such as Gossypium hirsutum cv. Sicala V-1, a cDNA library was constructed with mRNA isolated from root tissue of Sicala V-1, 24 h after inoculation with V. dahliae. The library was screened by a differential screening technique which was successful in identifying differences in gene expression between uninfected and V. dahliae-infected G. hirsutum root tissue. Among the differentially expressed clones, 51% represented up-regulated genes which had the potential to be involved in the defence response of G. hirsutum. The temporal expression patterns of nine suspected defence response genes were examined by northern blot analysis at several time intervals after inoculation with V. dahliae. The rapid increase in mRNA transcripts corresponding to each of these clones upon infection suggests a role for these genes in the defence response of G. hirsutum. Genes not previously associated with the defence response of the cotton plant, such as those for a 14-3-3-like protein and pathogenesis-related (PR) proteins, have been identified together with presumably novel genes, for which a definite function could not be ascribed.

RAPD (random amplified polymorphic DNA) markers generated by 30 random decamer primers were used to fingerprint 12 released cultivars and a breeding line of Gossypium hirsutum and 1 cultivar of G. barbadense presently under cultivation in Australia. Among a total of 453 developed markers, 69 (15.2%) were only present (unique) in the G. barbadense cultivar Pima S-7. Of the remaining markers, 128 (33.3%) were fixed in all 13 G. hirsutum cultivars. In pairwise comparisons of the degree of band sharing, nine closely-related cultivars showed 92.1-98.9% genetic similarity. Cluster analysis of genetic distance estimates between each of the cultivars revealed phylogenetic relationships in broad agreement with the known lineage of the cultivars. Ten of the G. hirsutum cultivars can be characterized individually based upon cultivar-specific RAPD markers, thus making it possible to differentiate closely related cultivars by molecular markers.

The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.

Nosocomial infections caused by Staphylococcus aureus strains resistant to methicillin and multiple antibiotics have reached epidemic proportions in Melbourne, Australia, over the past 5 years. Plasmid analysis of representative clinical isolates demonstrated the presence of three classes of plasmid DNA in most strains. Resistance to gentamicin, kanamycin, and tobramycin was usually mediated by an 18-megadalton plasmid but could also be encoded by a related 22-megadalton plasmid. Two distinguishable plasmids of 3 megadaltons each endowed resistance to chloramphenicol, and the third class consisted of small plasmids, each approximately 1 megadalton in size, with no attributable function. An extensive array of resistance determinants, including some which have usually been associated with a plasmid locus, were found to exist on the chromosome. Evidence that resistance to gentamicin, kanamycin, and tobramycin is chromosomally encoded in some clinical isolates suggests that this determinant may have undergone genetic translocation onto the staphylococcal chromosome.

The Staphylococcus aureus plasmid pSK1 carries Tn4001, a 4.7-kilobase (kb) transposon which specifies resistance to gentamicin, tobramycin, and kanamycin. In addition, pSK1 mediates resistance to trimethoprim and linked resistance to ethidium bromide (Ebr) and to quaternary ammonium compounds (Qar). Restriction endonuclease analysis of pSK1 and a deleted derivative of pSK1 revealed that the gene(s) responsible for Ebr Qar lies within a 5.2-kb HindIII fragment. This fragment has been cloned into the Escherichia coli plasmid vector pBR322, and transformants of an E. coli K-12 strain exhibited Ebr Qar. Subcloning of the 5.2-kb insert, combined with data from electron microscopic analysis of deleted derivatives of pSK1, located the Ebr Qar determinant(s) on a 2.3-kb segment of pSK1 DNA.

We describe a 4.5 kilobase transposon, Tn4001, which mediates resistance to gentamicin, tobramycin and kanamycin in Staphylococcus aureus. Originally detected in plasmid pSK1, Tn4001 was shown to undergo rec-independent transposition to the chromosome from this plasmid and from an inserted derivative of the plasmid pII147. Heteroduplexes between plasmids with and without Tn4001 demonstrated a characteristic stem and loop structure with inverted repeats of approx. 1.3 kilobases.

Resistance to gentamicin, tobramycin and kanamycin (GmrTmrKmr) in strains of Staphylococcus aureus isolated from clinical sources in Australia is mediated by a 4.7 kb transposable element, designated Tn4001. A 2.5 kb HindIII fragment which maps symmetrically within Tn4001, and encompasses the aminoglycoside-resistance coding region, has been shown to hybridise with fragments of identical size in HindIII digests of three different GmrTmrKmr plasmids, two of which were self-transmissible, from strains of S. aureus isolated in the USA. Examination by electronmicroscopy of self-annealed molecules of the North American GmrTmrKmr plasmids revealed the presence of stem and loop structures similar to those produced by Tn4001, but with shorter inverted repeats. These results suggest that GmrTmrKmr in strains of S. aureus isolated in the USA is, or once was, transposable, and that transposable elements analogous to Tn4001 may be found in isolates of GmrTmrKmr S. aureus worldwide.

Antibiotic multiresistant isolates of Staphylococcus aureus from outbreaks of nosocomial infection throughout Australia were found to possess essentially similar patterns of antibiotic resistance. Plasmid DNA profiles from these isolates exhibited a common pattern of large plasmids, of (15-22) X 10(6) mol. wt, associated with resistance to gentamicin, kanamycin and tobramycin, plasmids of 3 X 10(6) mol. wt, mediating resistance to chloramphenicol, and cryptic plasmids of 1 X 10(6) mol. wt. Restriction endonuclease digestion confirmed the presence of related plasmids in isolates from all the hospitals that were surveyed. The homogeneity of these organisms suggests the dissemination of a multiresistant, plasmid-bearing strain of S. aureus, or its derivatives, among geographically-separated hospitals in Australia.

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.

Strains of methicillin-resistant Staphylococcus aureus (MRSA), isolated from clinical sources in Victoria, were screened for the presence of plasmid DNA. Resistance to gentamicin, kanamycin, and tobramycin was found to reside on an 18-megadalton plasmid, and resistance to chloramphenicol was mediated by a 3-megadalton plasmid. No extra-chromosomal locus was found for resistance to several other antibiotics, including methicillin. Local strains of MRSA differ from their international counterparts by the possession of extensive chromosomal resistance determinants. Together with those encoded on plasmids, these determinants are capable of neutralising almost all available antistaphylococcal antibiotics.

The molecular and biochemical basis of resistance to high concentrations (MIC greater than or equal to 1000 mg/L) of trimethoprim (Tpr(H] was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(H) determinant (dfr A) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr(H) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(H) gene sequences among staphylococci.

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.

Molecular and genetic analysis of multiresistant isolates of Staphylococcus aureus from widely separated hospitals in Australia has demonstrated that these are clearly related, and that the predominant strains possess up to three different plasmids, which fall into the following classes: (i) small 1.6 kb plasmids, such as pSK3, which are phenotypically cryptic, (ii) 4.5 kb chloramphenicol resistance plasmids, such as pSK2, and (iii) the pSK1 family of multiresistance plasmids, which range in size from 20 to 42 kb and variously encode resistance to antiseptics and disinfectants, trimethoprim (Tpr), penicillin (Pcr) and the aminoglycosides gentamicin, tobramycin and kanamycin (Gmr Tmr Kmr). Gmr Tmr Kmr is encoded on the pSK1 family plasmids by transposon Tn4001, which was also detected on the chromosomes of some clinical isolates. Tn4001 is composed of inverted repeats of the insertion sequence IS256; these repeats flank a Gmr Tmr Kmr sequence encoding for a 57,000 dalton bifunctional protein with aminoglycoside acetyltransferase [AAC(6')] and phosphotransferase [APH(2")] activities. A Tn4001-like structure, which is defective in transposition but encodes for a Gmr Tmr Kmr determinant homologous with that on Tn4001, occurs on conjugative plasmids from strains isolated in North America. Physical studies indicate that Pcr, via a beta-lactamase, and Tpr, via a trimethoprim-insensitive dihydrofolate reductase (DHFR), are also encoded on the pSK1 family by transposons; these transposons have been designated Tn4002 and Tn4003, respectively. Tn4003 is flanked by direct repeats of the insertion sequence IS257. The evolution of the pSK1 family of multiresistance plasmids is traced through the transposition and genetic rearrangement of resistance determinants. Transposition and genetic rearrangement have also contributed to the evolution of a multiresistant chromosome in Staph. aureus. In the majority of contemporary multiply resistant Staph. aureus strains the determinants for resistance to erythromycin (Emr), fusidic acid, methicillin (Mcr), minocycline, rifampicin, spectinomycin, streptomycin, sulphonamides, tetracycline (Tcr), cadmium (Cdr), and mercury (Hgr) are chromosomally encoded; these strains also possess chromosomally encoded Pcr, via a beta-lactamase. Evidence indicates that some of these determinants, Pcr, Cdr, Hgr, and Tcr, were plasmid encoded in isolates collected from Australian hospitals prior to 1970. Through transposition and site-specific integration, they have since been acquired by the chromosome in more recent Staph. aureus strains.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.

The availability of recombinant DNA techniques has enabled the successful genetic analysis and manipulation of a range of C1 utilizing microorganisms. It has resulted in the identification of genes of interest on both plasmids and the chromosome; enabled the linkage of chromosomal genes to be determined; established the function and regulatory patterns of genes essential for utilization of C1 compounds and provided information on the evolution of methanogenic bacteria.

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.