Jiangella gansuensis gen. nov., sp nov., a novel actinomycete from a desert soil in north-west China

A novel actinomycete strain, designated YIM 002(T), was isolated from a desert soil sample in Gansu Province, north-west China. This actinomycete isolate formed well-differentiated aerial and substrate mycelia. In the early stages of growth, the substrate mycelia fragmented into short or elongated rods. Chemotaxonomically, it contained LL-2,6-diaminopimelic acid in the cell wall. The cell-wall sugars contained ribose and glucose. Phospholipids present were phosphatidylinositol mannosides, phosphatidylinositol and diphosphatidylglycerol. MK-9(H-4) was the predominant menaquinone. The major fatty acids were anteiso C-15:0 (35.92%), anteiso C-17:0 (15.84%), iso C-15:0 (10.40%), iso C-16:0 (7.07%) and C(17:10)w8c (9.37%). The G+C content of the DNA was 70 mol%. Phylogenetic analysis and signature nucleotide data based on 16S rRNA gene sequences showed that strain YIM 002(T) is distinct from all recognized genera of the family Nocardioidaceae in the suborder Propionibacterineae. On the basis of the phenotypic and genotypic characteristics, it is proposed that isolate YIM 002(T) be classified as a novel species in a new genus, Jiangella gansuensis gen. nov., sp. nov. The type strain is YIM 002(T) (= DSM 44835(T) = CCTCC AA 204001(T) = KCTC 19044(T)).

淡水湖泊中类蛭弧菌的分离鉴定与多样性分析

类蛭弧菌(BALOs)是一种微小的，能够高速运动的革兰氏阴性捕食细菌。它通过裂解宿主细胞来获得自己生长发育所需的物质、能量。类蛭弧菌分布范围广泛，土壤、海洋、湖泊、河流、下水道的污水、水管和水池之中都有它的存在。目前将与蛭弧菌具有相似生活特性的一类细菌称为 “类蛭弧菌”。 该研究从阿哈湖、百花湖和滇池三个内陆淡水湖泊中分离鉴定了宿主菌。利用分离得出的两株宿主菌分离培养类蛭弧菌。进行了16S rRNA基因序列分析。对不同富营养化的高原湖泊中类蛭弧菌的多样性进行比较。在以下几个方面取得了一些进展： 1. 改进了对类蛭弧菌的分离培养方法。我们直接从类蛭弧菌生活的环境中去寻找并提取宿主细菌。这样可以最大程度的保证实验室条件下类蛭弧菌的生长和真实环境中的相似性。我们的实验也证明了这种方法的有效性，并且在实验中我们获得了肠杆菌和黄色假单胞菌这两种类蛭弧菌的宿主菌。 2. 用新分离出来的两种宿主菌，通过噬菌斑生成、吸光度检测，PCR扩增等一系列实验证明我们成功的从淡水湖泊中分离得到了类蛭弧菌。 3、经过对所得的类蛭弧菌的16S rRNA基因序列进行检测,获得了不同类群的类蛭弧菌，并且还发现了新的类群。 3. 构建了分离的类蛭弧菌的系统发生树，对三个湖泊中类蛭弧菌的多样性进行了分析。

Classificacao taxonomica, baseada na caracterizacao molecular, das estirpes de rizobio recomendadas para a culturas da soja e do feijoeiro.

As culturas da soja [Glycine max (L.) Merril] e do feijoeiro (Phaseolus vulgaris) sao de grande importancia economica e social para o Brasil e ambas podem ser capazes de suprir suas necessidades do nutriente nitrogenio pela simbiose com bacterias da familia Rhizobiaceae. Para garantir a maximizacao do processo biologico, porem, deve-se proceder a inoculacao das sementes com estirpes de rizobio eficientes e competitivas, recomendadas pela pesquisa. No Brasil, sao comercializadas, anualmente, cerca de 13 milhoes de doses de inoculantes, sendo 99% destinadas a cultura da soja. Neste trabalho, determinou-se a posicao taxonomica das estirpes de rizobio recomendadas comercialmente para as duas culturas por duas tecnicas, o RFLP-PCR da regiao correspondente ao 16S rRNA (regiao conservada entre bacterias mas suficientemente variavel e carregando informacoes que permitem a determinacao das relacoes filogeneticas entre bacterias) e o sequenciamento parcial dos genes desta regiao. O sequenciamento parcial permitiu definir que duas das estirpes recomendadas para a cultura da soja, SEMIA 587 e SEMIA 5019, pertencem a especie Bradyrhizobium elkanii e as duas outras, SEMIA 5079 e SEMIA 5080, a especie B. japonicum. As duas estirpes recomendadas para a cultura do feijoeiro, SEMIA 4077 e SEMIA 4080, pertencem a especie Rhizobium tropici. As sequencias obtidas para essas estirpes foram depositadas no banco mundial de genes.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS: the suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). the ranking of the best single and combination of reference genes was determined by NormFinder, geNorm (TM), BestKeeper, and DataAssist (TM). in addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. the relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. the level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested. (C) 2013 Baishideng Publishing Group Co., Limited. All rights reserved.

A Phylogenetic Mixture Model for Detecting Pattern-Heterogeneity in Gene Sequence or Character-State Data

Mark Pagel, Andrew Meade (2004). A phylogenetic mixture model for detecting pattern-heterogeneity in gene sequence or character-state data. Systematic Biology, 53(4), 571-581. RAE2008

Bioinformatyczna analiza struktury i oddziaływań z substratami metylotransferaz RNA odpowiedzialnych za oporność bakterii na antybiotyki

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.

Comparative genomics of Bifidobacteria

Chapter 2 of this thesis describes the sequence analysis of 14 bifidobacterial genomes from various species of the genus Bifidobacterium, and the determination of their open pan-genome trend. This analysis first determined the total number of genes to be considered as the reservoir of functions available to representatives of this genus. Many identified genes are still uncharacterized, but may be involved in the adaptation to the gut environment. This comparative genomic analysis also determined a pool of ortholog functions used to infer their phylogenetic relationship, thereby providing a more robust approach compared to that based solely on the16S rRNA-encoding gene. The genome sequence of an isolate from the insect hindgut Bifidobacterium asteroides PRL2011 was fully characterized in Chapter 3, surprisingly revealing a putative respiratory metabolism, which was also found to be present in other insect isolates, suggesting that respiration was an ancient feature of this genus, but also an adaptative trait to different atmosferic oxygen levels. Chapter 4 of this thesis outlines a comparative study which focused on the analysis of representatives of the Bifidobacterium breve species, revealing that the genetic variability among members of this species principally consists of genes with a role in adaptation to host environment and gut colonization. Finally, Chapter 5 describes the analysis of the genome sequence of Bifidobacterium animalis subsp. lactis BLC1, a probiotic bacterium widely used in food industries as an ingredient of functional foods, providing information that will allow future investigations of this species.

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Co-orientation of replication and transcription preserves genome integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia.

BACKGROUND: West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease. METHODS: Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis. RESULTS: Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease. CONCLUSIONS: Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

The evolutionary origin of Indian Ocean tortoises (Dipsochelys).

Today, the only surviving wild population of giant tortoises in the Indian Ocean occurs on the island of Aldabra. However, giant tortoises once inhabited islands throughout the western Indian Ocean. Madagascar, Africa, and India have all been suggested as possible sources of colonization for these islands. To address the origin of Indian Ocean tortoises (Dipsochelys, formerly Geochelone gigantea), we sequenced the 12S, 16S, and cyt b genes of the mitochondrial DNA. Our phylogenetic analysis shows Dipsochelys to be embedded within the Malagasy lineage, providing evidence that Indian Ocean giant tortoises are derived from a common Malagasy ancestor. This result points to Madagascar as the source of colonization for western Indian Ocean islands by giant tortoises. Tortoises are known to survive long oceanic voyages by floating with ocean currents, and thus, currents flowing northward towards the Aldabra archipelago from the east coast of Madagascar would have provided means for the colonization of western Indian Ocean islands. Additionally, we found an accelerated rate of sequence evolution in the two Malagasy Pyxis species examined. This finding supports previous theories that shorter generation time and smaller body size are related to an increase in mitochondrial DNA substitution rate in vertebrates.

The Role of mRNA Translation in the Regulation of Ribosome Trafficking to the Endoplasmic Reticulum

The goal of this research is to identify the trafficking patterns that direct ribosomes to the endoplasmic reticulum (ER). It is widely believed that the SRP pathway is the only mechanism that cells use to localize mRNA and ribosomes to the ER, but this has been found not to be a sufficient explanation for the patterns of RNA localization in cells, namely that non-signal sequence-containing mRNA are translated on the ER and that ribosomes retain their membrane association after translation termination. First, a summary of the history of the field is presented to provide context for the key, unanswered questions in the field. Then, experiments employing [32Pi] pulse-chase labeling of HeLa cells over a time course to follow nascent ribosome trafficking are presented. The purpose of the cell labeling was to track rRNA processing and assembly into nascent ribosomes, followed by their export into the cytoplasm and recruitment into active polysomes. A detergent-based cell fractionation procedure was also utilized to separate the cytosol and ER compartments in order to observe ribosomes on their path as they exit the nucleus and either localize to the ER or cytosolic cellular compartment. Through this method, it was seen that ribosomes appear in both compartments at the same time, suggesting a mechanism may be occurring in addition to SRP-dependent ribosome trafficking. This research provides an understanding toward a mechanism that is not currently known, but will one day more fully explain the patterns of ribosomal localization.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Defining seasonal marine microbial community dynamics

Here we describe, the longest microbial time-series analyzed to date using high-resolution 16S rRNA tag pyrosequencing of samples taken monthly over 6 years at a temperate marine coastal site off Plymouth, UK. Data treatment effected the estimation of community richness over a 6-year period, whereby 8794 operational taxonomic units (OTUs) were identified using single-linkage preclustering and 21 130 OTUs were identified by denoising the data. The Alphaproteobacteria were the most abundant Class, and the most frequently recorded OTUs were members of the Rickettsiales (SAR 11) and Rhodobacteriales. This near-surface ocean bacterial community showed strong repeatable seasonal patterns, which were defined by winter peaks in diversity across all years. Environmental variables explained far more variation in seasonally predictable bacteria than did data on protists or metazoan biomass. Change in day length alone explains >65% of the variance in community diversity. The results suggested that seasonal changes in environmental variables are more important than trophic interactions. Interestingly, microbial association network analysis showed that correlations in abundance were stronger within bacterial taxa rather than between bacteria and eukaryotes, or between bacteria and environmental variables.