Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.The toxicity of phenolic compounds in the environment has fostered studies of bacteria that are able to tolerate and/or metabolize high levels of these compounds, particularly under anaerobic conditions (1, 4, 14, 21, 30, 36). Tannins are secondary polyphenolic compounds known primarily for their ability to bind to and precipitate proteins and other macromolecules. Tannins have been found in many habitats, including sewage sludge, forest litter, and the rumen (9, 14, 15, 28). Bacteria capable of degrading or tolerating tannins have been isolated from sewage sludge (14) and from the alimentary tracts of koalas (Phascolarctos cinereus) (33), goats (Capra hircus) (4, 30), and horses (Equus caballus) (31). Most of the isolates have been characterized phenotypically, and phylogenetic characterization has been limited to studies conducted in Australia (4, 34, 35) and Japan (31). Little is known about the geographic diversity and host species diversity of tannin-tolerant and tannin-degrading bacteria.The objective of this study was to characterize six recently isolated tannin-tolerant bacteria by examining their phenotypic characteristics and molecular phylogeny. These bacteria were isolated from the ruminal contents of goats (C. hircus), sheep (Ovis aries), white-tail deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni), all of which had consumed forage containing tannins. Our goal was to genetically and biochemically characterize tannin-tolerant bacteria isolated from different host animals in various geographic locations.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

Distribution of millipedes along an altitudinal gradient in the south of Lake Teletskoye,Altai Mts,Russia (Diplopoda)

The distribution of millipedes along an altitudinal gradient in the south of Lake Teletskoye, Altai, Russia based on new samples from the Kyga Profile sites, as well as on partly published and freshly revised material (Mikhaljova et al. 2007, 2008, 2014, Nefedieva and Nefediev 2008, Nefediev and Nefedieva 2013, Nefedieva et al. 2014), is established. The millipede diversity is estimated to be at least 15 species and subspecies from 10 genera, 6 families and three orders. The bulk of species diversity is confined both to low- and mid-mountain chern taiga forests and high-mountain shrub tundras, whereas the highest numbers, reaching up to 130 ind./m², is shown in subalpine Pinus sibirica sparse growths. Based on clustering studied localities on species diversity similarity two groups of sites are defined: low-mountain sites and subalpine sparse growths of Pinus sibirica ones.     

Genomic Plasticity of the Human Fungal Pathogen Candida albicans

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large-scale chromosome changes that arise in Candida albicans, and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest that C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate but also to generate large-scale genetic variation as a means of adaptation.C. albicans is a polymorphic yeast with a 16-Mb (haploid) genome organized in 8 diploid chromosomes (140, 154, 203). The C. albicans genome displays a very high degree of plasticity. This plasticity includes the types of genomic changes frequently observed with cancer cells, including gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity (reviewed in references 100, 117, and 157). Similar to somatic cancer cells, C. albicans reproduces primarily through asexual clonal division (65, 84). Nonetheless, it has retained much of the machinery needed for mating and meiosis (189), yet meiosis has never been observed (13, 120).C. albicans has two mating-type-like (MTL) alleles, MTLa and MTLα (76). The MTL locus is on the left arm of chromosome 5 (Chr5), approximately 80 kbp from the centromere. Most C. albicans isolates are heterozygous for the MTL locus, but approximately 3 to 10% of clinical isolates are naturally homozygous at MTL (104, 108). Mating can occur between strains carrying the opposite MTL locus, and most strains that were found to be naturally MTL homozygous are mating competent (104, 108). MTL-homozygous strains were also constructed from MTL-heterozygous strains by deletion of either the MTLa or MTLα locus (77) or by selection for Chr5 loss on sorbose (87, 115).Mating between these diploid strains of opposite mating type can occur both in vitro (115) and in vivo (77, 97). The products are tetraploid and do not undergo a conventional meiotic reduction in ploidy (12, 120). Rather, they undergo random loss of multiple chromosomes, a process termed “concerted chromosome loss,” until they reach a near-diploid genome content (2, 12, 53, 85). A subset of these cells also undergoes multiple gene conversion events reminiscent of meiotic recombination, and most remain trisomic for one to several chromosomes (53). While mating and concerted chromosome loss have been induced in the laboratory, the role of the parasexual cycle during the host-pathogen interaction and in the response to stresses, such as exposure to antifungal drugs, remains unclear. The prevailing model is that adaptive mutations (such as those that occur with the acquisition of drug resistance) evolve through somatic events, including point mutations, recombination, gene conversion, loss of heterozygosity, and/or aneuploidy (13).     

Delineation of a Carcinogenic Helicobacter pylori Proteome

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons ever develop cancer. The extensive genetic diversity inherent to this pathogen has precluded comprehensive analyses of constituents that mediate carcinogenesis. We previously reported that in vivo adaptation of a non-carcinogenic H. pylori strain endowed the output derivative with the ability to induce adenocarcinoma, providing a unique opportunity to identify proteins selectively expressed by an oncogenic H. pylori strain. Using a global proteomics DIGE/MS approach, a novel missense mutation of the flagellar protein FlaA was identified that affects structure and function of this virulence-related organelle. Among 25 additional differentially abundant proteins, this approach also identified new proteins previously unassociated with gastric cancer, generating a profile of H. pylori proteins to use in vaccine development and for screening persons infected with strains most likely to induce severe disease.Helicobacter pylori is a Gram-negative bacterial species that selectively colonizes gastric epithelium and induces an inflammatory response within the stomach that persists for decades (1, 2). Biological costs incurred by the long term relationship between H. pylori and humans include an increased risk for distal gastric adenocarcinoma (3–8), and eradication of this pathogen significantly decreases cancer risk among infected individuals without premalignant lesions (9). However, only a fraction of colonized persons ever develop neoplasia, and enhanced cancer risk is related to H. pylori strain differences, inflammatory responses governed by host genetic diversity, and/or specific interactions between host and microbial determinants (10).H. pylori strains are remarkably diverse (11–15), and the genetic composition of strains can change over time within an individual colonized stomach (16, 17). Despite this diversity, several genetic loci have been identified that augment disease risk. The cag pathogenicity island encodes a type IV bacterial secretion system, and the product of the terminal gene in this island, CagA, is translocated into host epithelial cells by the cag secretion system following adherence (18–20). Within the host cell, CagA undergoes Src- and Abl-dependent tyrosine phosphorylation (21) and activates the eukaryotic phosphatase SHP-2, leading to dephosphorylation of host cell proteins and cellular morphological changes (19–21). CagA also dysregulates β-catenin signaling (22, 23) and apical-junctional complexes (24), events linked to increased cell motility and oncogenic transformation in several models (25, 26). Another H. pylori constituent linked to gastric cancer is the cytotoxin VacA, encoded by the gene vacA, which is present in virtually all H. pylori strains (27). In vitro, VacA induces the formation of intracellular vacuoles (27) and can induce apoptosis (28), and vacuolating activity is significantly associated with the presence of the cag pathogenicity island (3).Approximately 20% of H. pylori bind to gastric epithelial cells in vivo (29), and sequence analysis has revealed that the H. pylori genome contains an unusually high number of ORFs relative to its genome size that are predicted to encode outer membrane proteins (15). BabA, a member of a family of highly conserved outer membrane proteins and encoded by the strain-specific gene babA2, binds the Lewis^b histo-blood group antigen on gastric epithelial cells (30, 31), and H. pylori babA2⁺ strains are associated with an increased risk for gastric cancer (30). However, not all persons infected with cag⁺ babA2⁺ toxigenic strains develop gastric cancer, indicating that additional H. pylori constituents are important in carcinogenesis.We recently identified a strain of H. pylori, 7.13, that reproducibly induces gastric cancer in two rodent models of gastritis, Mongolian gerbils and hypergastrinemic INS-GAS mice (22). This strain was derived via in vivo adaptation of a clinical H. pylori strain, B128, which induces inflammation, but not cancer, in rodent gastric mucosa. The oncogenic 7.13 phenotype is not due to an enhanced ability of strain 7.13 to colonize as there were no significant differences in gastric colonization density or efficiency between strains B128 and 7.13 as assessed by either quantitative culture or histology. However, carcinogenic strain 7.13 binds more avidly to gastric epithelial cells in vitro than does strain B128, suggesting that the two strains may variably express different outer membrane proteins.To define proteins that may mediate the development of H. pylori-induced gastric cancer, we performed two-dimensional (2D)¹ DIGE coupled with MS to identify differentially abundant membrane-associated and cytosolic proteins from non-carcinogenic H. pylori strain B128 and its carcinogenic derivative, strain 7.13 (22). DIGE/MS is a well established proteomics technology based on conventional 2D gel protein separations whereby prelabeling samples with spectrally resolvable fluorescent dyes and multiplexing samples onto a series of gels that contain a mixture of all experimental samples (internal standard) provide quantitative data on abundance changes for thousands of intact proteins from multiple experimental conditions, each measured in replicate for statistical confidence (32–36). Techniques including DIGE/MS have recently been utilized to robustly define differences in protein abundance profiles between bacterial strains and to compare expression patterns of proteins harvested from bacteria maintained under different growth conditions (37, 38).Utilizing DIGE/MS, we detected and identified 26 proteins with statistically significant differences between strains B128 and 7.13, including a novel cysteine-to-arginine mutation in the H. pylori flagellar protein FlaA. We demonstrate that this FlaA mutation results in structural and functional aberrations. Application of this technique to two genetically related bacterial strains that induce distinct phenotypes also identified several novel candidate H. pylori virulence factors, providing a framework for studies targeting the pathogenesis of microbially induced cancer.     

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.     

Isolation of Broad-Host-Range Replicons from Marine Sediment Bacteria

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888–895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the α and γ subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the γ-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5α and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.  The maintenance and horizontal transfer of extrachromosomal elements provide one mechanism by which microbial communities can rapidly adapt to changes in environmental conditions. This adaptation can be in the form of plasmid rearrangements and duplications (18, 40), a change in the plasmid copy number (40, 54), or lateral or horizontal movement of plasmids within bacterial populations. An example demonstrating the importance of plasmid-mediated genetic adaptation in natural microbial communities, likely caused by lateral transfer, is the increased frequencies (2- to 10-fold) of catabolic plasmids reported in bacterial isolates obtained from polluted marine and freshwater environments compared to isolates from nonpolluted or less impacted ecosystems (8, 23, 43). Plasmids also play a major role in promoting the widespread distribution of antibiotic resistance genes attributed to the intense and increased use of antibiotics (42).The ability of plasmids to self-transfer or to be mobilized by transfer-proficient plasmids and the ability to replicate in different bacterial hosts are key factors in the spread of plasmid-encoded genes within microbial communities. Plasmids which are considered to have broad host ranges in nature have the potential to significantly affect the microbial community structure and function due to their ability to replicate and be maintained in members of distantly related genera. Thus, to better understand gene flux in natural systems and hence the potential role of plasmids in promoting horizontal transfer within microbial communities, knowledge of the distribution, diversity, and host ranges of naturally occurring plasmids is necessary.At present, most indigenous plasmids from marine and freshwater systems have been only partially characterized with respect to host range, replication mechanisms, incompatibility groups, and conjugal abilities. Plasmids containing similar or related replication systems are considered incompatible if they cannot coexist in a host cell (12, 41). This trait has facilitated the grouping of plasmids from gram-negative bacteria, mainly members of the family Enterobacteriaceae, into more than 30 different incompatibility groups (3). While molecularly based plasmid classification or replicon typing by using DNA sequences of replication origins and incompatibility loci of well-characterized plasmids has been useful in classifying plasmids from bacterial isolates of medical importance (9, 10, 14), plasmids from various marine microbial communities, including sediments, biofilms, bulk water, and the marine air-water interface, have been recently shown to contain incompatibility and replication regions unrelated to those currently defined (11, 53).The present study was undertaken to characterize, at the molecular level, the replication and incompatibility loci of naturally occurring plasmids isolated from gram-negative marine heterotrophs for use as replicon probes to classify and type, at the molecular level, plasmids present in bacterial populations of marine sediments. Replication origins were obtained from plasmids ranging in size from 6 to 30 kb isolated from culturable bacteria of coastal California marine sediments (53). Phylogenetic analysis indicated that the plasmids were initially isolated from bacteria belonging to the α and γ-3 subclasses of the class Proteobacteria. Although a sequence and hybridization analysis of the replication origins from the marine plasmids confirmed the lack of homology with previously described plasmids, the replication regions contained features commonly found in previously characterized plasmid replication origins. The replication origins of the naturally occurring plasmids appear to have a broad host range, as indicated by their ability to replicate in members of diverse gram-negative marine genera. In addition to molecular characterization of the indigenous plasmids, the persistence of the replicons in marine sediment bacterial populations was determined by PCR amplification of microbial community DNA extracted on different dates and examined for the presence of homologous plasmid replication sequences.     

Phylogenetic Analysis Indicates Evolutionary Diversity and Environmental Segregation of Marine Podovirus DNA Polymerase Gene Sequences

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase (pol) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA pol gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus pol sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.Marine viruses are the most abundant (41) and diverse (2, 6) biological entities in the ocean. They affect community composition by causing the lysis of specific subsets of the microbial community (22, 28, 46, 47) and, by killing numerically dominant host taxa, may influence species evenness and richness (24, 28, 43, 50). Despite the abundance of bacteriophages in marine systems and their important roles in marine microbial composition, little is known about the distribution and diversity of specific groups of marine viruses. However, most marine bacteriophage isolates are tailed phages (3) belonging to the order Caudovirales (27), which comprises the families Myoviridae, Podoviridae, and Siphoviridae.Podoviruses are classified into several groups (e.g., T7-like, P22-like, and phi-29-like) based on genome size, genome arrangement, and shared genes and can be readily isolated from seawater (11, 16, 42, 45). Genomic analysis of roseophage SIO1 (33), cyanophage P60 (7), vibriophage VpV262 (21), and cyanophage PSSP7 (40) suggests that many of the isolates are T7-like. Despite the apparently wide distribution of podoviruses in the sea, and their potential importance as agents of microbial mortality, there has been little effort to explore their diversity.Sequence analysis of representative genes is one approach that has been used to examine the genetic diversity of specific groups of marine viruses. For example, homologues for structural genes (g20 and g23) found in T4-like phages are found in some marine myoviruses (18, 20) and have been used to examine the distribution, diversity, and evolutionary relationships among marine myoviruses (12, 14, 17, 37, 38, 49). Other studies have used DNA polymerase (pol) to examine the diversity of viruses infecting eukaryotic phytoplankton (8, 38) and have shown that phylogenies constructed with this gene are congruent with established viral taxonomy (9, 36, 37).Although it is not universally present, family A DNA pol is a good target for examining the diversity of podoviruses (4). Our study presents a newly designed set of PCR primers that amplify a longer fragment of the DNA polymerase from a much larger suite of podoviruses and shows that the diversity within marine podoviruses as revealed by DNA pol sequences is far greater than previously realized.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]