Approaches to investigating the ecology of plasmids in marine bacterial communities

To better understand prokaryotic gene flux in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of plasmid populations in marine bacterial communities, information on the distribution, diversity, and ecological traits of marine plasmids is necessary. This mini-review highlights recent insights gained into the molecular diversity and ecology of plasmids occurring in marine microbial communities.     

Progress towards understanding the nature of chromatid breakage

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.     

Mechanisms determining the fate of dispersed bacterial communities in new environments

Recent work has shown that dispersal has an important role in shaping microbial communities. However, little is known about how dispersed bacteria cope with new environmental conditions and how they compete with local resident communities. To test this, we implemented two full-factorial transplant experiments with bacterial communities originating from two sources (freshwater or saline water), which were incubated, separately or in mixes, under both environmental conditions. Thus, we were able to separately test for the effects of the new environment with and without interactions with local communities. We determined community composition using 454-pyrosequencing of bacterial 16S rRNA to specifically target the active fraction of the communities, and measured several functional parameters. In absence of a local resident community, the net functional response was mainly affected by the environmental conditions, suggesting successful functional adaptation to the new environmental conditions. Community composition was influenced both by the source and the incubation environment, suggesting simultaneous effects of species sorting and functional plasticity. In presence of a local resident community, functional parameters were higher compared with those expected from proportional mixes of the unmixed communities in three out of four cases. This was accompanied by an increase in the relative abundance of generalists, suggesting that competitive interactions among local and immigrant taxa could explain the observed ‘functional overachievement''. In summary, our results suggest that environmental filtering, functional plasticity and competition are all important mechanisms influencing the fate of dispersed communities.     

Progress towards a molecular-level structural understanding of amyloid fibrils

The problem of determining and understanding the molecular structures of amyloid fibrils has attracted considerable attention and effort over the past several years. Although complete, high-resolution structures have not yet been obtained, key features of protein and peptide conformations and supramolecular organization within amyloid fibrils have been elucidated using a variety of novel experimental methods, including magnetic resonance spectroscopies, electron microscopy, X-ray and neutron scattering, and biochemical techniques. The experimental data are beginning to shed light on issues such as the nature of the intermolecular interactions that stabilize amyloid structures, the molecular structural basis for polymorphism in amyloid fibrils, the universality of amyloid structures, and the balance between structural order and disorder within amyloid fibrils. Recent structural data will contribute to an improved understanding of the mechanisms of amyloid formation and to the development of therapeutic agents for amyloid diseases.     

Progress towards the understanding and control of sugar beet rhizomania disease

Rhizomania is a soil-borne disease that occurs throughout the major sugar beet growing regions of the world, causing severe yield losses in the absence of effective control measures. It is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the obligate root-infecting parasite Polymyxa betae . BNYVV has a multipartite RNA genome with all natural isolates containing four RNA species, although some isolates have a fifth RNA. The larger RNA1 and RNA2 contain the housekeeping genes of the virus and are always required for infection, whereas the smaller RNAs are involved in pathogenicity and vector transmission. RNA5-containing isolates are restricted to Asia and some parts of Europe, and these isolates tend to be more aggressive. With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through the use of resistant cultivars. A single dominant resistance gene, Rz1 , has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance. More recently, new variants of BNYVV have evolved (both with and without RNA5) that are able to cause significant yield penalties on resistant cultivars. These isolates are not yet widespread, but their appearance has resulted in accelerated searches for new sources of resistance to both the virus and the vector. Combined virus and vector resistance, achieved either by conventional or transgenic breeding, offers the sugar beet industry a new approach in its continuing struggle against rhizomania.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly

The microsomal triglyceride transfer protein (MTP) is necessary for the proper assembly of the apolipoprotein B containing lipoproteins, very low density lipoprotein and chylomicrons. Recent research has significantly advanced our understanding of the role of MTP in these pathways at the molecular and cellular level. Biochemical studies suggest that initiation of lipidation of the nascent apolipoprotein B polypeptide may occur through a direct association with MTP. This early lipidation may be required to allow the nascent polypeptide to fold properly and therefore avoid ubiquitination and degradation. Concerning the addition of core neutral lipids in the later stages of lipoprotein assembly, cell culture studies show that MTP lipid transfer activity is not required for this to occur for apolipoprotein B-100 containing lipoproteins. Likewise, MTP does not appear to directly mediate addition of core neutral lipid to nascent apoB-48 particles. However, new data indicate that MTP is required to produce triglyceride rich droplets in the smooth endoplasmic reticulum which may supply the core lipids for conversion of nascent, dense apoB-48 particles to mature VLDL. In addition, assembly of dense apolipoprotein B-48 containing lipoproteins has been observed in mouse liver in the absence of MTP. As a result of these new data, an updated model for the role of MTP in lipoprotein assembly is proposed.     

Progress towards understanding disease mechanisms in small vertebrate models of neuronal ceroid lipofuscinosis

Model systems provide an invaluable tool for investigating the molecular mechanisms underlying the NCLs, devastating neurodegenerative disorders that affect the relatively inaccessible tissues of the central nervous system. These models have enabled the assessment of behavioural, pathological, cellular, and molecular abnormalities, and also allow for development and evaluation of novel therapies. This review highlights the relative advantages of the two available small vertebrate species, the mouse and zebrafish, in modelling NCL disease, summarising how these have been useful in NCL research and their potential for the development and testing of prospective disease treatments. A panel of mouse mutants is available representing all the cloned NCL gene disorders (Cathepsin D, CLN1, CLN2, CLN3, CLN5, CLN6, CLN8). These NCL mice all have progressive neurodegenerative phenotypes that closely resemble the pathology of human NCL. The analysis of these models has highlighted several novel aspects underlying NCL pathogenesis including the selective nature of neurodegeneration, evidence for glial responses that precede neuronal loss and identification of the thalamus as an important pathological target early in disease progression. Studies in mice have also highlighted an unexpected heterogeneity underlying NCL phenotypes, and novel potential NCL-like mouse models have been described including mice with mutations in cathepsins, CLC chloride channels, and other lysosome-related genes. These new models are likely to provide significant new information on the spectrum of NCL disease. Information on NCL mice is available in the NCL Mouse Model Database (). There are homologs of most of the NCL genes in zebrafish, and NCL zebrafish models are currently in development. This model system provides additional advantages to those provided by NCL mouse models including high-throughput mutational, pharmacogenetic and therapeutic technique analyses. Mouse and zebrafish models are an important shared resource for NCL research, offering a unique possibility to dissect disease mechanisms and to develop therapeutic approaches.     

Phenotypic plasticity in bacterial plasmids

Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments.     

Molecular structure of bacterial plasmids

[This corrects the article on p. 369 in vol. 36.].     

Regulation of the replication of bacterial plasmids

This is a survey of the available data on the structure of the replication apparatus and the control mechanisms of bacterial plasmids replication. All plasmids are classified as having three types of replicons according to the mechanisms of replication initiation. The role of plasmid--determined positive and negative regulatory elements involved in controlling the number of copies and incompatibility is discussed.     

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10⁻⁵ to 10⁻⁸ per recipient. A total of 12 distinct plasmids, designated pB1–pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10⁻¹ to 10⁻² per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPβ plasmids at the DNA sequence level. In order to characterize the gene “load” of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified. Received: 11 October 1999 / Accepted: 11 January 2000     

Rains, drains and active strains: towards online assessment of wastewater bacterial communities

Wastewater treatment is one of the largest scale and arguably the most commercially important biotechnological process in the world. Bacterial breakdown of waste materials facilitates the safe disposal of effluents into receiving water bodies. Given this significance, research has focused on identifying the keystone species on which efficient treatment is based. However, unravelling the microbial diversity within such systems has proven difficult. This is highlighted by our lack of detailed knowledge of the microbial interactions within these complex populations, limiting our ability to fully exploit bacterial degradative abilities. Even with the incorporation of new emerging molecular techniques, there have been no investigations linking genetic sequence to microbial function and successful treatment operation. To reach this goal, researchers need the ability to identify, enumerate and monitor the metabolic functions of subpopulations within these complex bacterial communities. Flow cytometry (FCM) combined with fluorescence-based molecular identification techniques provides a method for such studies. Moreover, single-cell sorting provides a unique opportunity to identify and remove individual cells of interest. Laboratory culture of sorted cells is often possible and permits the use of more traditional microbiological techniques to backup molecular investigations. Utilising this approach will advance our understanding of wastewater treatment processes and help maintain and enhance plant operation to improve efficiency.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.