Macromolecular Synthesis and Degradation in Arthrobacter During Periods of Nutrient Deprivation

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.     

Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

Due to sampling difficulties, little is known about microbial communities associated with sinking marine snow in the twilight zone. A drifting sediment trap was equipped with a viscous cryogel and deployed to collect intact marine snow from depths of 100 and 400 m off Cape Blanc (Mauritania). Marine snow aggregates were fixed and washed in situ to prevent changes in microbial community composition and to enable subsequent analysis using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). The attached microbial communities collected at 100 m were similar to the free-living community at the depth of the fluorescence maximum (20 m) but different from those at other depths (150, 400, 550, and 700 m). Therefore, the attached microbial community seemed to be “inherited” from that at the fluorescence maximum. The attached microbial community structure at 400 m differed from that of the attached community at 100 m and from that of any free-living community at the tested depths, except that collected near the sediment at 700 m. The differences between the particle-associated communities at 400 m and 100 m appeared to be due to internal changes in the attached microbial community rather than de novo colonization, detachment, or grazing during the sinking of marine snow. The new sampling method presented here will facilitate future investigations into the mechanisms that shape the bacterial community within sinking marine snow, leading to better understanding of the mechanisms which regulate biogeochemical cycling of settling organic matter.     

Estimating Bacterial Production in Marine Waters from the Simultaneous Incorporation of Thymidine and Leucine

We examined the simultaneous incorporation of [³H]thymidine and [¹⁴C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 ± 0.2 [mean ± standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 ± 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (<5 h). With the dual-label method we found that thymidine and leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts.     

Bacterial Diversity in Shallow Oligotrophic Marine Benthos and Overlying Waters: Effects of Virus Infection,Containment, and Nutrient Enrichment

Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate–based sediments of Florida Bay (USA). Sediment–water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to 201 cm sediment depth enclosing 80 L of water. Mesocosms were amended each day for 14 d with 10 µM NH ₄ ⁺ and 1 µM PO ₄ ^3– . In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 ± 3) was higher than in the water column (148 ± 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources.     

Diurnal Variation of Cell Proliferation in Three Bacterial Taxa from Coastal North Sea Waters

Pulse-labeling with bromodeoxyuridine (BrdU) in combination with fluorescence in situ hybridization was applied to quantify the percentage of proliferating cells in coastal North Sea waters. In order to assess diurnal variability, we sampled eight or nine times, respectively, within 3 consecutive days at two seasons. Bacteria affiliated with the Roseobacter, SAR86, and NOR5 lineages constituted on average 19% ± 3%, 8% ± 2%, and 6% ± 1% of all cells in May 2002 and 17% ± 3%, 10% ± 2%, and 11% ± 3% in August. The relative abundances of the three populations either remained stable, or they changed very gradually during the observation periods. On average, 38 and 39% of all Bacteria exhibited DNA de novo synthesis in May and August, respectively. The fractions of proliferating cells in bacteria of the SAR86 (May, 59%; August, 72%) and the Roseobacter (48 and 53%) lineages were significantly above the community average. A substantial cell proliferation of population NOR5 (34%) was only encountered in August, concomitant with a dinoflagellate bloom. Significant short-term fluctuations of DNA-synthesizing cells were observed in Roseobacter during May and in NOR5 during August, hinting at a pronounced (temporal or spatial) mesoscale patchiness of growth rates in these populations. Since the BrdU proliferation assay is susceptible to misinterpretation, we also modeled the expected number of labeled cells at increasing BrdU incubation times in a slowly growing bacterial population. We suggest that the absence of visible DNA synthesis in marine bacterioplankton cells after DNA pulse-labeling must not be interpreted as an indication of cell “inactivity.”     

Enhanced Arginine Methylation of Programmed Cell Death 4 Protein during Nutrient Deprivation Promotes Tumor Cell Viability

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.     

Characterization of Klebsiella Isolates from Natural Receiving Waters and Comparison with Human Isolates

Two hundred sixty-six strains of Klebsiella pneumoniae isolated from natural water sources in geographically diverse areas (Florida, Massachusetts, and Oregon) were analyzed to determine the serotype, biochemical, virulence, and antimicrobial susceptibility differences between these natural strains and human Klebsiella isolates. Sixty of 72 defined serotypes were found among 210 typable strains. Geographic patterns were present, but in general were not pronounced among serotypes. Reactions with 28 biochemical tests showed percentage responses which were very similar to the summaries of primarily human Klebsiella isolates (as reported by Edwards and Ewing, 1972) and that represented diverse geographic sampling. Virulence studies in representative strains showed no geographic variability and little difference from comparable hospital patient-obtained isolates. In contrast to human hospital isolates, strains demonstrated 90% or greater susceptibility to all antibiotics except ampicillin and carbenicillin; and in further contrast, there was little multiple antibiotic resistance beyond that with ampicillin and carbenicillin.     

Effects of Light and CO on the Survival of a Marine Ammonium-Oxidizing Bacterium during Energy Source Deprivation

The chemolithotrophic ammonium-oxidizing bacterium Nitrosomonas cryotolerans responds uniquely to nutrient deprivation by lowering its endogenous respiration and anabolic processes to undetectable levels during starvation, thus appearing to enter a dormant state. To ascertain whether this state protects the cells from further stresses (as seen with endospore-forming bacteria), the starved cells were subjected to two known inhibitors, CO and light. It was found that long-term-starved cells were less resistant than freshly starved cells to light inhibition. Both long-term-starved cells and freshly starved cells were unaffected by CO.     

焦化废水处理系统中不同培养基分离的细菌种群多样性

以焦化废水处理系统的微生物群落为对象,对3种不同培养基（YPG、LB、WW）分离细菌的能力及分离物的种群多样性组成进行了比较研究。同一悬浮污泥样品在YPG、LB和WW培养基上的活菌计数结果分别为1.6×10.6 CFU/mL、7.0×10.5 CFU/mL和98×10.5CFU/mL。从每种培养基10-4稀释度平板上共分离137株分离物。将所有分离物扩增近全长的16S rDNA并用限制性内切酶HinfI对PCR产物进行ARDRA（Amplified rDNA restriction analysis）多态性分析,共得到14种不同的操作分类单元（Operational Taxonomic Unit, OUT）。其中YPG培养基上的分离物显示了8种不同的OTUs,而WW培养基和LB培养基分离物只分别显示了6种和4种OTUs。YPGOTU1和WWOTU6所包含的菌株分别占到总分离物的30%和22.3%,为优势分离物。ERICPCR基因组指纹图分析表明,前者的34株分离物共有20种不同的指纹图类型,而后者的25株分离物只有3种。因此,就分离焦化废水处理系统中的细菌及对分离物进行种群多样性的研究而言,YPG培养基比其他两种培养基更合适。     

Small Changes in pH Have Direct Effects on Marine Bacterial Community Composition: A Microcosm Approach

As the atmospheric CO₂ concentration rises, more CO₂ will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15–8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.     

Brain Monoamine Changes in Rats After Short Periods of Ozone Exposure

We have shown in our laboratory that cat's and rat's sleep disturbances are produced by 24 h of ozone (O₃) exposure, indicating that the central nervous system is affected by this gas. To demonstrate the probable changes in brain neurotransmitters, we evaluated the monoamine contents of the midbrain and striatum of rats exposed to 1 part per million O₃ for 1 or 3 hours periods. The results were compared with rats exposed to fresh air and to those exposed to 3 hours of O₃ followed by 1 or 3 hours of fresh air. We found a significant increase in dopamine (DA) and its metabolites noradrenaline (NA) and 3,4 dihydroxyphenylacetic acid (DOPAC), as well as an increase in the 5-hydroxyindolacetic acid (5-HIAA) contents of the striatum. There were no changes in homovanillic acid (HVA) and serotonin (5-HT) levels during O₃ exposure. Additionally, an increase in DA, NA and 5-HIAA in the midbrain during O₃ exposure was observed. Turnover analysis revealed that DA increased more than its metabolites in both the midbrain and striatum. However, the metabolite of 5-HT, i.e. 5-HIAA, increased more than its precursor, this reaching statistical significance only in the midbrain. These findings demonstrate that O₃ or its reaction products affect the metabolism of major neurotransmitter systems as rapidly as after 1 h of exposition.     

Changes in Ultrastructure and Subunit Composition of Protein Body in Rice Endosperm during Germination

Changes in ultrastructure of protein bodies in subaleurone cells of rice endosperm during germination were studied by transmission electron microscopy. The subaleurone cells contained two different types of protein bodies: PB-I (spherical) and PB-II (crystalline). Both types of protein bodies were deconstructed during germination. But there was a considerable difference in digestibility between PB-I and PB-II. PB-II which did not have a dense core was easily digested from the central portion when germination began. At 6 days of germination, PB-II was almost deconstructed. On the other hand, PB-I which displayed concentric rings with a dense core was digested from the outside after 3 days of germination. At 9 days of germination, many kernels of the spherical protein bodies remained.

Changes in subunit composition of protein bodies during germination were investigated by SDS-polyacrylamide gel electrophoresis. Protein body fractions were isolated from germinating grains at various stages by enzymatic digestion and two-phase system, then subjected to SDS-polyacrylamide gel. As germination proceeded, 15 (b₁), 20 (d₁), 24 (e), 35 (f₁) and 37 (f₃) kdaltons subunits decreased. On the other hand, 16 (b₂), 21 (d₂) and 36 (f₂) k daltons subunits remained at the later stage of germination. We think that PB-I contains b₂, d₂ and f₂ subunits and is attacked only from the outside at middle and later stages of germination by de novo protease. On the contrary, PB-II contains b₁ d₁ e, f₁ and f₃ subunits is utilized at an early stage of germination. PB-II may possibly contain latent protease. The breakdown process of PB-I was by exo-type digestion, on the contrary, that of PB-II was by endo-type digestion.     

Composition and Activity of Marine Alkane-Degrading Bacterial Communities in the Transition from Suboxic to Anoxic Conditions

The impact of the oxygen supply rate (OSR) on the metabolic activity and on the composition of hexadecane-degrading bacterial communities in a quasi-anoxic milieu (nominal DOT=0%) was studied in continuous cultures containing intertidal sediment. The dilution rate was kept constant at 0.035 h⁻¹. The OSR was stepwise reduced from 3.5 mmol O₂L⁻¹ h⁻¹ to 0.06 mmol O₂L⁻¹ h⁻¹. Activity was determined by analyzing the respiration quotient (RQ) and the rates of hexadecane degradation (Q_Hex), of hexadecane mineralization, and of protein production (PPR). The community composition and size were investigated by fluorescence in situ hybridization (FISH), by dilution plating (colony forming units or CFU), and by most probable number (MPN). The culture showed an aerobic hexadecane metabolism down to an OSR of 0.35 mmol O₂L⁻¹ h⁻¹. Below this OSR, anaerobic metabolism was initiated. The relationship among the RQ, PPR, Q_Hex, and the OSR can be approximated by hyperbola (Michaelis-Menten kinetics). We suggest that the metabolic adaptation of the culture to low OSRs is due to regulation of protein expression and enzyme activity. Reducing the OSR resulted in minor but significant changes in the concentration of different physiological and phylogenetic groups. This means that, in addition to protein expression and activity regulation, the adaptation of the population to low OSRs is due to changes in the community composition.     

Surviving Chytridiomycosis: Differential Anti-Batrachochytrium dendrobatidis Activity in Bacterial Isolates from Three Lowland Species of Atelopus

In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25°C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.     

Changes in Membrane Fluidity and Protein Composition during Release of Cucumber Seeds from Dormancy by a Higher Temperature Shift

Dormancy of seeds of cucumber (Cucumis sativus L.) was inducedby imbibing in -1.8 MPa polyethylene glycol 6000 (PEG) solutionand pulsing with far red light for 15 min prior to washing anddrying. When re-imbibed with water at 20 °C, dormancy wasbroken by raising the temperature to 30 °C for 6 h. Thistreatment was also effective when -0.9 MPa PEG was present duringre-imbibition and high temperature. Seeds with broken dormancywere found to germinate in water over a smaller temperaturerange than seeds in which dormancy had not been induced. Whenthe duration of the temperature shift to 30 °C was varied,germination percentage increased from 7 to 60% after 6 h, butlonger exposures up to 12 h had no further promoting effect.The time course of germination after transfer to water following6 h at 30 °C in PEG showed piercing of the perisperm-endospermenvelope after 9–12 h and radicle protusion after 12–15h. If PEG was retained after high temperature treatment no visiblegermination was observed. Thus, to study membrane fluidity andthe protein content associated with germination, seeds weresampled 9 h after high temperature treatment. To study the germinablebut not germinating state, seed held in PEG for 9 h rather thanin water was used. Dormant seed was sampled before the hightemperature treatment. Membrane fluidity was assessed usingfluorescence polarization of membrane fractions treated withDPH (1,6-diphenyl-1,3,5-hexatriene) or its derivatives. Membraneproteins were compared using one-dimensional SDS-PAGE electrophoresis.Intracellular membrane fluidity was not increased in the transitionfrom the dormant to germinable state, but did increase in thetransition to germination. There were no detected changes inintracellular membrane proteins during either transition. Inplasma membrane fractions, fluidity increased during both transitions,while a marked increase in 21, 18 and 17 kD proteins was observedin the transition from germinable to germinating state. Thusmodification of plasma membrane fluidity rather than changesin protein profile is associated with the high temperature releaseof cucumber seeds from dormancy. Copyright 2000 Annals of BotanyCompany     

Diversity of Bacterial Isolates from Commercial and Homemade Composts

The diversity of heterotrophic bacterial isolates of three commercial and two homemade composts was studied. The commercial composts were produced from poultry litter (PC), sewage sludge (SC), municipal solid waste (MC), and homemade composts (thermal compost [DC] and vermicompost [VC]) from food wastes. The taxonomic and physiological diversity of the heterotrophic culturable bacteria was assessed using phenotypic and genotypic characterization and the analysis of the partial 16S rRNA gene sequence. Composts DC and SC presented the higher genotypic diversity, as could be inferred from the number of distinct genotypic patterns observed, 28 and 21, respectively. Gram-positive bacteria, mainly Firmicutes, were predominant in all the composts. Some organisms related with taxa rarely reported in composts, as Rhodanobacter spathiphylli, Moraxella osloensis, Lysobacter, Corynebacterium, Pigmentiphaga kullae, and new taxa were also isolated. The highest relative proportion of isolates able to degrade starch was found in compost SC (>70%), to degrade gelatine in compost DC (>70%), to degrade Tween 80 in compost PC (>90%), and to degrade poly-epsilon-caprolactones in compost DC (>80%). Compost MC presented the lowest relative proportions of isolates able to degrade starch (<25%), gelatine (<20%), and poly-epsilon-caprolactone (<40%). When compared with the others, the homemade composts presented higher relative proportions of Gram-positive isolates able to inhibit the target organisms Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, or Pseudomonas aeruginosa. In compost MC, none of the Gram-positive isolates was able to inhibit those targets.     

Effects of Dissolved Organic Matter Photoproducts and Mineral Nutrient Supply on Bacterial Growth in Mediterranean Inland Waters

Sunlight reacts with dissolved organic matter (DOM) modifying its availability as bacterial substrate. We assessed the impact of DOM photoproducts and mineral nutrient supply on bacterial growth in seven inland waters from the South of Spain, where DOM is characterized by low chromophoric content and long residence time. Factorial experiments were performed with presence vs absence of DOM photoproducts and mineral nutrient supply. In six of the seven experiments, we found a significant and negative effect of DOM photoproducts on bacterial growth and a significant and positive effect of mineral nutrient supply. The interaction of these two factors leaded to a compensation of negative effects of photoproducts by availability of mineral nutrients. Dissolved organic matter diagenetic status and the ionic environment where organic carbon is dissolved can be influencing bacterial DOM processing.     

The Effects of Dietary Ferric Iron and Iron Deprivation on the Bacterial Composition of the Mouse Intestine

The influence of dietary ferric iron on the intestinal microbiota of mice was investigated with a view to promoting benign lactic acid bacteria (which have minimal iron requirements) in order to enhance colonization-resistance potential. Three groups of eight mice received a diet differing only in iron content, for a period of 12 weeks. Dietary iron deprivation resulted in overall increased small intestinal bacterial populations, including lactic acid bacteria, but these differences were generally not significant (p > 0.05). With the exception of coliforms, all examined bacterial groups (anaerobes, micro-aerophiles, lactobacilli, and enterococci) were significantly (p < 0.05) elevated in the colons of iron-deprived mice. The relatively low numbers of total anaerobes in the colons of iron-replete and iron-overloaded mice suggested that, as well as promotion of bacteria under iron-deprived condition, provision of ferric iron suppressed bacteria, probably by oxidation of normally reduced environments. Received: 13 October 2000 / Accepted: 20 December 2000