Influence of fungal-bacterial interactions on bacterial conjugation in the residuesphere

Conjugal gene transfer among bacteria in the residuesphere (area between decaying plant material and soil) of leaves of barley straw was studied. The residuesphere was shown to be a hot-spot for conjugal gene transfer compared to conjugation in sterile sand and non-sterile bulk soil. Impact of fungal colonisation of the residuesphere on bacterial colonisation and conjugation was also investigated. The inhibition of fungal colonisation, due to the application of an eukaryotic inhibitor, increased bacterial colonisation of the residuesphere in soil microcosms compared to non-treated leaves. This treatment also had a transient, positive effect on conjugation. Bacterial conjugation in the residuesphere of leaves subjected to 17 days of fungal colonisation was significantly lower than in the residuesphere of non-colonised leaves. Fungal biomass, as measured by chitinase activity, was inversely related to the conjugation efficiency.     

A classification scheme for mobilization regions of bacterial plasmids

Transmissible plasmids can be classified according to their mobilization ability, as being conjugative (self-transmissible) or mobilizable (transmissible only in the presence of additional conjugative functions). Naturally occurring mobilizable plasmids carry the genetic information necessary for relaxosome formation and processing, but lack the functions required for mating pair formation. Mobilizable plasmids have a tremendous impact in horizontal gene transfer in nature, including the spread of antibiotic resistance. However, analysis of their promiscuity and diversity has attracted less attention than that of conjugative plasmids. This review will focus on the analysis of the diversity of mobilizable plasmids. For this purpose, we primarily compared the amino acid sequences of their relaxases and, when pertinent, we compared these enzymes with conjugative plasmid relaxases. In this way, we established phylogenetic relationships among the members of each superfamily. We conducted a database and literature analysis that led us to propose a classification system for small mobilizable plasmids in families and superfamilies according to their mobilization regions. This review outlines the genetic organization of each family of mobilization regions, as well as the most relevant properties and relationships among their constituent encoded proteins. In this respect, the present review constitutes a first approach to the characterization of the global gene pool of mobilization regions of small mobilizable plasmids.     

Community-based degradation of 4-chorosalicylate tracked on the single cell level

4-Chlorosalicylate (4-CS) can be degraded completely by a bacterial consortium consisting of Pseudomonas reinekei (MT1), Achromobacter spanius (MT3) and Pseudomonas veronii (MT4). The fourth species Wautersiella falsenii (MT2) is thought to act as a 'necrotizer' of the community. Single cell approaches were used to follow every species' degradation activity within the community by assuming that growth and proliferation are activity markers for the utilization of 4-CS and its degradation pathway intermediates as carbon and energy sources. A primary/secondary antibody staining technique for species differentiation was applied and a species-resolved determination of proliferation activity by flow cytometry undertaken. Degradation was followed by quantifying 4-CS and the resulting intermediates by HPLC. A good correlation of HPLC bulk data with the proliferation activity states of every species within the community was found. It was also assumed that reduced activity of strain MT4 and increased proliferation of strain MT2 might have caused an observed breakdown of the consortium grown in the bioreactor. The double staining technique provided the chance to follow bacterial cell states and their roles in mixed cultures without applying labelled substrates. It is therefore in line with single cell techniques already successfully applied in biotechnology for developing strategies to optimize microbially catalyzed production processes.     

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat _GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10⁻⁷ and 4.7 × 10⁻⁷ transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10⁻⁵ transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA ⁺ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future. Received: 22 April 1997 / Accepted: 4 November 1997     

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.     

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.     

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.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

Inhibition of bacterial segregation by early functions of phage Mu and association of replication protein B with the inner cell membrane

Summary Infection of Mu-sensitive bacteria with a recombinant phage that carries the EcoRI·C fragment from the immunity end of wild type Mu DNA causes filamentous growth. Transmission electron microscopy revealed that the cell-division cycle was inhibited at, or prior to, the initiation of septation. The filamentation does not occur after infection of Mu-immune bacteria or after infection with a phage carrying the same EcoRI·C fragment, but with an IS1 insertion in gene B of Mu, showing that either gpB and/or some non-essential functions (e.g. kil) mapping downstream from the insertion are required for the inhibition of cell division. These data and previously published evidence suggest that in the killing of E. coli K12 by early Mu functions expressed from the cloned EcoRI·C fragment, two components have to be distinguished: one, a highly efficient elimination of plasmid DNA carrying the early Mu genes, and second, a series of interactions with host functions conducent to an inhibition of cell division. It is suggested that functions normally involved in the SOS reaction participtate in the inhibition of cell division by early Mu functions. Infected bacteria synthesize the replication protein B (M_R 33000) of Mu, which was found by cell fractionation experiments to be associated with the inner cell membrane. The role of this association for filamentous growth and for the integrative replication of the phage is discussed. The recombinant phage might be useful as a tool for the study of the E. coli cell division cycle.     

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.     

A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments

Bacterial microcompartments (MCPs) are protein‐based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high‐throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP‐targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP‐targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways.     

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.     

Electrostatic role of the non-heme iron complex in bacterial photosynthetic reaction center

To elucidate the role of the non-heme iron complex (Fe-complex) in the electron transfer (ET) events of bacterial photosynthetic reaction centers (bRC), we calculated redox potentials of primary/secondary quinones Q(A/B) (E(m)(Q(A/B))) in the Fe-depleted bRC. Removing the Fe-complex, the calculated E(m)(Q(A/B)) are downshifted by approximately 220 mV/ approximately 80 mV explaining both the 15-fold decrease in ET rate from bacteriopheophytin (H(A)(-)) to Q(A) and triplet state occurrence in Fe-depleted bRC. The larger downshift in E(m)(Q(A)) relative to E(m)(Q(B)) increases the driving-energy for ET from Q(A) to Q(B) by 140 meV, in agreement with approximately 100 meV increase derived from kinetic studies.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B_A), bacteriopheopytin (H_A), the primary quinone (Q_A) to the secondary quinone (Q_B). Although the non-heme iron complex (Fe complex) is located between Q_A and Q_B, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q_A and Q_B in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q_A/B (E_m(Q_A/B)) and the Fe complex (E_m(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E_m(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E_m(Fe) downshift by 230 mV upon formation of Q_A⁻ (but not Q_B⁻) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Potentialities of mermithid nematodes for the biocontrol of blackflies (Diptera: Simuliidae)--a review

Mermithids comprise a family of nematodes which invariably kill/sterilize their insect host(s). These nematodes have considerable potential as biocontrol agents of agricultural insect pests and medically important insect vectors. More specifically, mermithid nematodes appear to regulate natural population of blackflies. The taxonomy of the Mermithidae has been only partially evaluated and the taxonomic status of many representatives is uncertain. At least three mermithid genera and species parasitize North American blackflies, although a more varied mermithid fauna probably exists. The host specificity of mermithid parasites of simuliids is variable, but these nematodes do not appear to infect other stream fauna. The sporadic distribution of mermithid parasites of simuliids among potential biotopes may be associated with a relatively inefficient mode of dispersal for such nematodes. Detailed information is lacking concerning stages in the life cycles of these pathogens and their synchronization with the simuliid host. Mermithids cause pathogenic effects upon several blackfly tissues, although no information is available concerning physiological manifestations of mermithid parasitism in blackflies. A brief review of the present state of knowledge of simuliid taxonomy and bionomics is presented. The physiology of blackflies and their mermithid parasites has been largely ignored. The potentialities of mermithid nematodes for the biocontrol of blackflies are assessed and a feasible research programme presented, in relation to the present state of knowledge of mermithid-simuliid interrelationships and related areas of insect nematology.