The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

The ultrastructure of Macropodinium moiri and revised diagnosis of the Macropodiniidae (Litostomatea : Trichostomatia)

The ultrastructural features of Macropodinium moiri were investigated. The somatic cortex is composed of two lateral non-ciliated zones covered with trapezoidal plates and separated by a trough-like dorsoventral groove (DVG) which divides the cell into left and right halves. The somatic kineties occupy the margins of the DVG and are composed of monokinetids whose infraciliature shows a typical litostome pattern. The pellicular plates are lamellate, and separated by V-shaped grooves which are lined by thick-walled vacuoles. The DVG cortex is composed of electron-opaque U-shaped ribs which alternate with electron-lucent saccular structures. The DVG surface is composed of small regular pellicular sacs built up to form the ridges of the dorsal DVG. The vestibulum forms a laterally compressed cone with left/right differentiation. The basal section of its non-ciliated right side is internally lined (outer to innermost) by longitudinal fibres, nematodesmata and transverse microtubular ribbons. The left side bears the vestibular kineties and in its basal section is lined (outer to innermost) by small nematodesmata and transverse tubules. Cytoplasmic organelles include endoplasmic reticulum, starch granules and a single contactile vacuole surrounded by patches of nephridioplasm. Hydrogenosomes are absent and coccoid Gram-positive bacteria lie under the ciliated portions of the cell. This set of characteristics differs significantly from those of the all other trichostomes; Macropodiniidae is therefore designated Trichostomatia incertae sedis. A revised familial diagnosis of the Macropodiniidae is proposed.

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.

The ultrastructure of Amylovorax dehorityi comb. nov and erection of the Amylovoracidae fam. nov (Ciliophora : Trichostomatia)

The ultrastructural features of the holotrichous ciliates inhabiting macropodid maruspials were investigated to resolve their morphological similarity to other trichostome ciliates with observed differences in their small subunit rRNA gene sequences. The ultrastructure of Amylovorax dehorityi nov. comb. (formerly Dasytricha dehorityi) was determined by transmission electron microscopy. The somatic kineties are composed of monokinetids whose microtubules show a typical litostome pattern. The somatic cortex is composed of ridges which separate kinety rows, granular ectoplasm and a basal layer of hydrogenosomes lining the tela corticalis. The vestibulum is an invagination of the pellicle lined down one side with kineties (invaginated extensions of the somatic kineties); transverse tubules line the surface of the vestibulum and small nematodesmata surround it forming a cone-like network of struts. Cytoplasmic organelles include hydrogenosomes, irregularly shaped contractile vacuoles surrounded by a sparse spongioplasm, food vacuoles containing bacteria and large numbers of starch granules. This set of characteristics differs sufficiently from those of isotrichids and members of the genus Dasytricha to justify the erection of a new genus (Amylovorax) and a new family (Amylovoracidae). Dasytricha dehorityi, D. dogieli and D. mundayi are reassigned to the new genus Amylovorax and a new species A. quokka is erected. While the gross morphological similarities between Amylovorax and Dasytricha may be explained by convergent evolution, ultrastructural features indicate that these two genera have probably diverged independently from haptorian ancestors by successive reduction of the cortical and vestibular support structures.

New arsenite-oxidizing bacteria isolated from Australian gold mining environments - Phylogenetic relationships

Nine novel arsenite-oxidizing bacteria have been isolated from two different gold mine environments in Australia. Four of these organisms grow chemolithoautotrophically with oxygen as the terminal electron acceptor, arsenite as the electron donor, and carbon dioxide-bicarbonate as the sole carbon source. Five heterotrophic arsenite-oxidizing bacteria were also isolated, one of which was found to be both phylogenetically and physiologically identical to the previously described heterotrophic arsenite oxidizer misidentified as Alcaligenes faecalis. The results showed that this strain belongs to the genus Achromobacter. Phylogenetically, the arsenite-oxidizing bacteria fall within two separate subdivisions of the Proteobacteria. Interestingly, the chemolithoautotrophic arsenite oxidizers belong to the alpha-Proteobacteria, whereas the heterotrophic arsenite oxidizers belong to the beta-Proteobacteria.

The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition

Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.

Transformation processes, pathways, and possible sources of distinctive polychlorinated dibenzo-p-dioxin signatures in sink environments

In recent years, studies on environmental samples with unusual dibenzo-p-dioxin (PCDD) congener profiles were reported from a range of countries. These profiles, characterized by a dominance of octachlorinated dibenzodioxin (OCDD) and relatively low in dibenzofuran (PCDF) concentrations, could not be attributed to known sources or formation processes. In the present study, the processes that result in these unusual profiles were assessed using the concentrations and isomer signatures of PCDDs from dated estuarine sediment cores in Queensland, Australia. Increases in relative concentrations of lower chlorinated PODS and a relative decrease of OCDD were correlated with time of sediment deposition. Preferred lateral, anaerobic dechlorination of OCDD represents a likely pathway for these changes. In Queensland sediments, these transformations result in a distinct dominance of isomers fully chlorinated in the 1,4,6,9-positions (1,4-patterns), and similar 1,4-patterns were observed in sediments from elsewhere. Consequently, these environmental samples may not reflect the signatures of the original source, and a reevaluation of source inputs was undertaken. Natural formation of PCDDs, which has previously been suggested, is discussed; however, based on the present results and literature comparisons, we propose an alternative scenario. This scenario hypothesizes that an anthropogenic PCDD precursor input (e.g. pentachlorophenol) results in the contamination. These results and hypothesis imply further investigations are warrented into possible anthropogenic sources in areas where natural PCDD formation has been suggested.

Pea mania: deranged behaviour in cattle grazing a pea crop (Pisum sativum var arvense)

Hypersensitivity to external stimuli, progressing in some animals to manic behaviour, occurred in a cattle herd that grazed a crop of field peas (Pisum sativum var arvense) in the pre-flowering stage. Haematological and biochemical analyses eliminated hypomagnesaemia and ketosis as diagnoses. Other than two steers euthanased due to injuries sustained during manic episodes, all affected animals survived, recovering over 3 days when moved to alternative pasture. No necropsies were conducted. No microbial pathogens or endophytes were found on or in the plants. A previously reported incident in Victoria in 1987 in cattle grazing peas appeared to be of a similar nature. Environmental factors leading to these incidents were not clearly identified.

Tracing the fate of N-15-enriched feed in an intensive shrimp system

The fate of N-15-nitrogen-enriched formulated feed fed to shrimp was traced through the food web in shallow, outdoor tank systems (1000 1) stocked with shrimp. Triplicate tanks containing shrimp water with and without sediment were used to identify the role of the natural biota in the water column and sediment in processing dietary nitrogen (N). A preliminary experiment demonstrated that N-15-nitrogen-enriched feed products could be detected in the food web. Based on this, a 15-day experiment was conducted. The ammonium (NH4+) pool in the water column became rapidly enriched (within one day) with N-15-nitrogen after shrimp were fed N-15-enriched feed. By day 15, 6% of the added N-15-nitrogen was in this fraction in the 'sediment' tanks compared with 0.4% in the 'no sediment' tanks. The particulate fraction in the water column, principally autotrophic nanoflagellates, accounted for 4-5% of the N-15-nitrogen fed to shrimp after one day. This increased to 16% in the 'no sediment' treatment, and decreased to 2% in the 'sediment' treatment by day 15. It appears that dietary N was more accessible to the phytoplankton community in the absence of sediment. The difference is possibly because a proportion of the dietary N was buried in the sediment in the 'sediment' treatment, making it unavailable to the phytoplankton. Alternatively, the dietary N was retained in the NH4+ pool in the water column since phytoplankton growth, and hence, N utilization was lower in the 'sediment' treatment. The lower growth of phytoplankton in the 'sediment' treatment appeared to be related to higher turbidity, and hence, lower light availability for growth. The percentage N-15-nitrogen detected in the sediment was only 6% despite the high capacity for sedimentation of the large biomass of plankton detritus and shrimp waste. This suggests rapid remineralization of organic waste by the microbial community in the sediment resulting in diffusion of inorganic N sources into the water column. It is likely that most of the dietary N will ultimately be removed from the tank system by water discharges. Our study showed that N-15-nitrogen derived from aquaculture feed can be processed by the microbial community in outdoor aquaculture systems and provides a method for determining the effect of dietary N on ecosystems. However, a significant amount of the dietary N was not retained by the natural biota and is likely to be present in the soluble organic fraction. (C) 2002 Elsevier Science B.V. All rights reserved.

Distributional profiles of homologous open reading frames among bacterial phyla: implications for vertical and lateral transmission

If open reading frames (ORFs) have been transmitted primarily by vertical descent, the distributional profile of orthologues of each ORF should be congruent with the organismal tree or a subtree thereof. Distributional patterns not reconciled parsimoniously with tree-like descent and loss are prima facie evidence of lateral gene transfer. Herein, a rigorous criterion for recognizing ORF distributions is described and implemented; it does not require the inference of phylogenetic trees, nor does it assume any specific tree. Because lineage-specific differences in rates of sequence change can also generate unexpected distributional patterns, rate artefacts, were controlled for by requiring pairwise matches between ORFs to exceed a rigorous inclusion threshold, but absence of a match was assessed against a more-permissive exclusion threshold. Applying this dual-threshold criterion to cross-domain and cross-phylum distributional patterns for ORFs in 23 bacterial genomes, a relative abundance of ORFs was observed that find a match in exactly seven other bacterial phyla; 94-99% of these ORFs also find matches among the Archaea and/or Eukarya. In the larger (and some smaller) bacterial genomes, ORFs that find matches in exactly one other bacterial phylum are also relatively abundant, but fewer of these have non-bacterial homologues; most of their matches within the Bacteria are to the Proteobacteria and/or Firmicutes, which cannot be sister lineages to all bacteria. ORFs that are neither distributed universally among the Bacteria, nor necessarily shared with topologically adjacent lineages, are preferentially enriched in large bacterial genomes.

The structural and functional diversity of naturally occurring antimicrobial peptides

Antimicrobial peptides occur in a diverse range of organisms from microorganisms to insects, plants and animals. Although they all have the common function of inhibiting or killing invading microorganisms they achieve this function using an extremely diverse range of structural motifs. Their sizes range from approximately 10-90 amino acids. Most carry an overall positive charge, reflecting a preferred mode of electrostatic interaction with negatively charged microbial membranes. This article describes the structural diversity of a representative set of antimicrobial peptides divided into five structural classes: those with agr-helical structure, those with bgr-sheet structure, those with mixed helical / bgr- sheet structure, those with irregular structure, and those incorporating a macrocyclic structure. There is a significant diversity in both the size and charge of molecules within each of these classes and between the classes. The common feature of their three-dimensional structures is, however, that they have a degree of amphipathic character in which there is separate localisation of hydrophobic regions and positively charged regions. An emerging trend amongst antimicrobial proteins is the discovery of more macrocyclic analogues. Cyclisation appears to impart an additional degree of stability on these molecules and minimizes proteolytic cleavage. In conclusion, there appear to be a number of promising opportunities for the development of novel clinically useful antimicrobial peptides based on knowledge of the structures of naturally occurring antimicrobial molecules.

delta N-15 values of tropical savanna and monsoon forest species reflect root specialisations and soil nitrogen status

A large number of herbaceous and woody plants from tropical woodland, savanna, and monsoon forest were analysed to determine the impact of environmental factors (nutrient and water availability, fire) and biological factors (microbial associations, systematics) on plant delta(15)N values. Foliar delta(15)N values of herbaceous and woody species were not related to growth form or phenology, but a strong relationship existed between mycorrhizal status and plant delta(15)N. In woodland and savanna, woody species with ectomycorrhizal (ECM) associations and putative N-2-fixing species with ECM/arbuscular (AM) associations had lowest foliar delta(15)N values (1.0-0.6parts per thousand), AM species had mostly intermediate delta(15)N values (average +0.6parts per thousand), while non-mycorrhizal Proteaceae had highest delta(15)N values (+2.9 to +4.1parts per thousand). Similar differences in foliar delta(15)N were observed between AM (average 0.1 and 0.2parts per thousand) and non-mycorrhizal (average +0.8 and +0.3parts per thousand) herbaceous species in woodland and savanna. Leguminous savanna species had significantly higher leaf N contents (1.8-2.5% N) than non-fixing species (0.9-1.2% N) indicating substantial N acquisition via N-2 fixation. Monsoon forest species had similar leaf N contents (average 2.4% N) and positive delta(15)N values (+0.9 to +2.4parts per thousand). Soil nitrification and plant NO3- use was substantially higher in monsoon forest than in woodland or savanna. In the studied communities, higher soil N content and nitrification rates were associated with more positive soil delta(15)N and plant delta(15)N. In support of this notion, Ficus, a high NO3- using taxa associated with NO3- rich sites in the savanna, had the highest delta(15)N values of all AM species in the savanna. delta(15)N of xylem sap was examined as a tool for studying plant delta(15)N relations. delta(15)N of xylem sap varied seasonally and between differently aged Acacia and other savanna species. Plants from annually burnt savanna had significantly higher delta(15)N values compared to plants from less frequently burnt savanna, suggesting that foliar N-15 natural abundance could be used as marker for assessing historic fire regimes. Australian woodland and savanna species had low leaf delta(15)N and N content compared to species from equivalent African communities indicating that Australian biota are the more N depauperate. The largest differences in leaf delta(15)N occurred between the dominant ECM Australian and African savanna (miombo) species, which were depleted and enriched in N-15, respectively. While the depleted delta(15)N of Australian ECM species are similar to those of previous reports on ECM species in natural plant communities, the N-15-enriched delta(15)N of African ECM species represent an anomaly.