Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: Revealed by differential display

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics, A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22 alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.

Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. fulciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (ACC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2(.)5 x 10(-9) mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and antimalarial drug therapy by demonstrating that even with lon mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

Latitudinal variability in symbiont specificity within the widespread scleractinian coral Plesiastrea versipora

We examined the genetic diversity of symbiotic dinoflagellates (Symbiodinium sp.) in the widespread hermatypic coral Plesiastrea versipora from tropical/subtropical (north-eastern Australia) and temperate waters (south-eastern Australia) using restriction fragment length polymorphisms of partial 18S ribosomal DNA (rDNA), together with sequence analysis of partial 28S rDNA. This study revealed that P. versipora associates with at least two distinct genotypes of symbiotic dinoflagellates and that the presence of these genotypes varies with latitude. P. versipora colonies from subtropical and tropical waters contained symbionts belonging to Symbiodinium clade C, while P. versipora colonies at high-latitude sites contained clade B. Variability within the two groups of symbionts (clades H and C) was minimal, suggesting possible host fidelity. The geographically distinct varieties of symbionts within the tissue of this hermatypic coral are likely to be associated with algal physiological differences, which in turn may relate to changing selective pressures as a function of latitude along the eastern Australian seaboard.

Genetic variability of the symbiotic dinoflagellates from the wide ranging coral species Seriatopora hystrix and Acropora longicyathus in the Indo-West Pacific

The scleractinian coral species, Seriatopora hystrix and Acropora longicyathus, are widely distributed throughout the latitudinal range of the tropical west Pacific. These 2 coral species live in a mutually beneficial relation with symbiotic dinoflagellates (zooxanthellae), which are passed to their progeny by vertical transmission (zooxanthellate eggs or larvae) and horizontal transmission (eggs or larvae that acquire symbionts from the environment), respectively. For S. hystrix, vertical transmission might create biogeographically isolated and genetically differentiated symbiont populations because the extent of its larval migration is known to be limited. On the other hand, horizontal transmission in corals such as A. longicyathus may result in genetically connected symbiont populations, especially if its zooxanthellae taxa are widely distributed. To examine these hypotheses, symbionts were collected from colonies of S. hystrix and A. longicyathus living in the Great Barrier Reef (Australia), South China Sea (Malaysia) and East China Sea (Ryukyus Archipelago, Japan), and were examined using restriction fragment length polymorphism and sequence analysis of large and small subunit rRNA genes. Phylogenetic analysis assigned the symbionts to 1 of 3 taxonomically distinct groups, known as clades. Symbionts from Australian and Japanese S. hystrix were placed in Clade C, and Malaysian S. hystrix symbionts in the newly described Clade D. Seven of 11 Australian and all Japanese and Malaysian colonies of A. longicyathus had symbiotic dinoflagellates that also grouped with Clade C, but symbionts from the remaining Australian colonies of A. longicyathus grouped with Clade A. Analysis of molecular variance of Clade C symbionts found significant genetic variation in 1 or more geographic groups (69.8%) and to a lesser extent among populations within geographic regions (13.6%). All populations of Clade C symbionts from S. hystrix were genetically differentiated according to geographic region. Although Clade C symbionts of A. longicyathus from Japan resolved into a distinct geographic group, those from Australia and Malaysia did not and were genetically connected. We propose that these patterns of genetic connectivity correlate with differences in the dispersal range of the coral or symbiont propagules and are associated with their respective modes of symbiont transmission.

Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci of P. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289-296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types

Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified, Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates,vith the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class, The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.

A spongin-boring alpha-proteobacterium is the etiological agent of disease in the Great Barrier Reef sponge Rhopaloeides odorabile

High levels of mortality in the Mediterranean bath sponge industry have raised concerns for the future of sponge farms. Healthy sponges feed predominantly on bacteria, and many harbour a wide diversity of inter- and extra-cellular symbiotic bacteria. Here we describe the first isolation and description of a pathogenic bacterium from an infected marine sponge. Microbiological examination of tissue necrosis in the Great Barrier Reef sponge Rhopaloeides odorabile resulted in isolation of the bacterial strain NW4327. Sponges infected with strain NW4327 exhibited high levels of external tissue necrosis, and the strain was re-isolated from infected sponges. A single morphotype, which had burrowed through the collagenous spongin fibres causing severe necrosis, was observed microscopically. Strain NW4327 was capable of degrading commercial preparations of azo-collagen, providing further evidence of its involvement in spongin fibre necrosis, Strain NW4327 disrupted the microbial community associated with R. odorabile and was able to infect and kill healthy sponge tissue. 16S rRNA sequence analysis revealed that strain NW4327 is a novel member of the alpha-proteobacteria.

Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.

Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula)

Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three. loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes.

Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development

Blepharophimosis ptosis epicanthus inversus syndrome (BPES) is a human disorder caused by mutations in the forkhead transcription factor gene FOXL2 and is characterized by facial dysmorphology combined in some cases with ovarian failure. To better understand the role of FOXL2 in the etiology of ovarian failure in BPES, we examined its expression in embryonic ovaries of mice, chickens, and red-eared slider turtles, representatives of three phylogenetically distant vertebrate groups that have different mechanisms of sex determination. Expression of Foxl2 was detected in early ovaries of all three species around the time of sex determination and was associated with both somatic and germ cell populations in mice. Expression was sexually dimorphic in all cases. Sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. FoxL2 was found to be autosomal in chickens, and therefore unlikely to represent the dominant ovarian-determining gene that has been postulated to exist as a possible explanation for female heterogamety in birds. Our observations suggest that BPES may result from early abnormalities in regulating the development of the fetal ovary, rather than premature degeneration of the postnatal or adult ovary. Further, our results suggest that FOXL2 is a highly conserved early regulator of vertebrate ovarian development.

Lactobacillus ruminis is a predominant lactic acid producing bacterium in the caecum and rectum of the pig

Aims: To identify the predominant lactic acid producing bacteria in the small intestine, caecum and the rectum of the healthy pig. Methods and Results: Samples obtained from the large intestine of healthy pigs post-mortem were cultured using a modified agar-MRS medium in roll tubes. Thirteen isolates were selected on the basis of their morphological characteristics and Gram stain reaction for gene sequencing. These isolates were characterized by DNA sequence analysis of 16S rDNA. Eight isolates were identified as Lactobacillus ruminis , two as Enterococcus faecium , one as Mitsuokella multiacidus and two as Escherichia coli . Conclusion: This is the first report of Lact. ruminis as the dominant lactic acid bacteria in the large intestine of the pig. Significance and Impact of the Study: The results suggest that Lact. ruminis is a dominant bacterium in the large intestine of the healthy pig. Future work should focus on the role of this bacterium in relation to the physiological function of the intestine and the health of the animal.