Individual Physiological Adaptations Enable Selected Bacterial Taxa To Prevail during Long-Term Incubations.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures.IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle effect") may occur due to the enclosure itself. In this study, we analyzed the bacterial communities that originated from three different salinities of the Baltic Sea, comparing their compositions and physiological activities both at the early stage and after 55 days of incubation. Our results suggested that internal carbon storage strategies impact the success of certain bacterial species, independent of the experimental manipulation. Thus, while enclosure experiments remain valid tools in environmental research, microbial community composition shifts must be critically followed. This investigation of the metaproteome during long-term batch enclosures expanded our current understanding of the so-called "bottle effect," which is well known to occur during enclosure experiments.

Detecting genetic structure in migrating bowhead whales off the coast of Barrow, Alaska.

We develop a general framework for analysing and testing genetic structure within a migratory assemblage that is based on measures of genetic differences between individuals. We demonstrate this method using microsatellite DNA data from the Bering-Chukchi-Beaufort stock of bowhead whales (Balaena mysticetus), sampled via Inuit hunting during the spring and autumn migration off Barrow, Alaska. This study includes a number of covariates such as whale ages and the time separation between captures. Applying the method to a sample of 117 bowhead whales, we use permutation methods to test for temporal trends in genetic differences that can be ascribed to age-related effects or to timing of catches during the seasons. The results reveal a pattern with elevated genetic differences among whales caught about a week apart, and are statistically significant for the autumn migration. In contrast, we find no effects of time of birth or age-difference on genetic differences. We discuss possible explanations for the results, including population substructuring, demographic consequences of historical overexploitation, and social structuring during migration.

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

Cold induction of the Bacillus subtilis bkd operon is mediated by increased mRNA stability.

Recently it has been demonstrated that the ptb - bcd - buk - lpdV - bkdAA - bkdAB - bkdB operon ( bkdoperon) of Bacillus subtilis, which encodes the enzymes that catalyze the degradation of branched-chain amino acids, is inducible by a temperature downshift from 37 to 18 degrees C. Deamination and oxidative decarboxylation of isoleucine generates 2-methyl-butyryl-CoA, which serves as the precursor of anteiso-branched fatty acid species. Most probably, the induction of this operon upon cold shock ensures an increase in the content of anteiso-branched fatty acids in the membrane lipids at low temperature, thus permitting maintenance of membrane fluidity at lower temperatures. In the present study, we have analyzed the mechanism of cold induction of the bkd operon and of four further cold-inducible transcriptional units in B. subtilis. We demonstrate that cold induction of these genes is mediated by an increase in the stability of the corresponding mRNAs. None of the promoters that control the five transcriptional units analyzed is actually cold-inducible. Furthermore, the results of this study indicate that the 5' leader regions are not involved in the cold-induced stabilization of the mRNAs. The structural elements that enhance mRNA stability must therefore be restricted to the 3'-ends and/or the coding regions.

Electric chips for rapid detection and quantification of nucleic acids.

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli.

The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background.

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7.

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.

Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.

Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes.

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis. The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10. The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Suicide rates from 1960 to 1989 in Norwegian physicians compared with other educational groups.

The aim of the present study is to compare suicide rates between 1960 and 1989 for Norwegian physicians with corresponding rates for other Norwegians with and without university education, by age, gender, and five-year period, based on death certificates for all Norwegians who died in the period 1960-1989. There were 82 registered physician suicides, of which 9 were female, 265 suicides by persons with other university education, and 11,165 by persons with no university education. Suicide rate is measured in number of deaths per 100,000 person years. Crude suicide rates were 47.7 (95% CI 37.7-60.4) for male physicians, 20.1 (17.7-22.9) for other male university graduates, and 22.7 (22.2-23.2) for men with no university education. The corresponding figures for females were 32.3 (15.8-63.7), 13.0 (8.4-19.8) and 7.7 (7.5-8.0). Both for males and females, suicide rates, controlled for age and period, were significantly higher for physicians than for persons with other or no university education. Poisson modelling showed that the risk of suicide for male physicians has the same age pattern as for other males with higher education. In 1985-89 the suicide rate for male physicians increased nearly linearly from about 35 at the age 35-40 to about 100 at the age 75-79, which was almost three times higher than for the other male university graduates. For the age group 50-54 the estimated rate increases from about 50 in 1960-64 to about 90 in 1985-89. For the female physicians, the low number of cases prevents reliable estimation of trends. For male physicians, the trend from 1960 to 1989 is increasing. The estimated risk for a single physician to commit suicide was almost 5 times that of a married or co-habitant colleague. For 52% of the male and 85% of the female physicians the suicide method was poisoning. This is about twice the rates in the general population.

Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins.

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg. g(-1)) compared to the fusion protein SpA-betagalactosidase (138 mg. g(-1)) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the 1st order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg. g(-1) to 120 mg. g(-1). The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg. g(-1) in 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.

Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations.

The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein alpha-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed on the transcriptional and the translational levels. After the induction of the tac-regulated overexpression of the recombinant model protein, a significant but transient increase of the mRNA levels of the heat shock genes lon and dnaK could be observed. The mRNA level of the gene coding for the inclusion body-associated protein IbpB showed the strongest increase and remained at a clearly higher level until the end of the fermentation. By contrast, the mRNA levels of htrA and ppiB were decreased after induction of the alpha-glucosidase overexpression. Analysis of the soluble cytoplasmic protein fraction 3 h after induction revealed increased levels of the chaperones GroEL, DnaK, and Tig and a decrease in the protein levels of the two ribosomal proteins S6 and L9, the peptidylprolyl-cis-trans-isomerase PpiB, and the sigma(38)-dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the alpha-glucosidase inclusion bodies. N-terminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous alpha-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the alpha-glucosidase protein aggregates.

Regulation of the expression of the cold shock proteins CspB and CspC in Bacillus subtilis.

The small acidic proteins CspB and CspC are the major cold shock-induced proteins of Bacillus subtilis. Analysis of mRNA revealed a transient four-fold increase in the transcription level of both genes during cold shock. The cspB and cspC mRNAs are dramatically stabilised after a temperature downshift from 37 degrees C to 15 degrees C. The data in this study support the idea that the expression of CspB and CspC in B. subtilis during cold shock is regulated mainly at the post-transcriptional level, as is also the case with CspA in Escherichia coli.

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Analysis of the expression and function of the sigmaB-dependent general stress regulon of Bacillus subtilis during slow growth.

Glucose-limited continuous cultures were used to analyze sigmaB activity at decreasing growth rates. Expression of the sigmaB-dependent genes gsiB and ctc started to increase at a growth rate of 0.2 h-1, and both genes were induced approximately fivefold at a growth rate of 0.1 h-1 as compared to expression at the maximal growth rate. However, maximal sigmaB activity was only reached when the growth stopped as a result of the exhaustion of the carbon and energy source glucose. During glucose-limited growth, increased expression of the general stress regulon at growth rates below 0.2 h-1 did not provide wild-type cells with a growth advantage over sigB mutants. Instead, expression of the stress regulon seems to constitute a significant burden during glucose-limited growth, resulting in a selective growth advantage of the sigB mutant as compared to the wild-type at a growth rate of 0.08 h-1.

Simulated likelihood methods for complex double-platform line transect surveys.

The conventional line transect approach of estimating effective search width from the perpendicular distance distribution is inappropriate in certain types of surveys, e.g., when an unknown fraction of the animals on the track line is detected, the animals can be observed only at discrete points in time, there are errors in positional measurements, and covariate heterogeneity exists in detectability. For such situations a hazard probability framework for independent observer surveys is developed. The likelihood of the data, including observed positions of both initial and subsequent observations of animals, is established under the assumption of no measurement errors. To account for measurement errors and possibly other complexities, this likelihood is modified by a function estimated from extensive simulations. This general method of simulated likelihood is explained and the methodology applied to data from a double-platform survey of minke whales in the northeastern Atlantic in 1995.

Hazard models for line transect surveys with independent observers.

The likelihood function for data from independent observer line transect surveys is derived, and a hazard model is proposed for the situation where animals are available for detection only at discrete time points. Under the assumption that the time points of availability follow a Poisson point process, we obtain an analytical expression for the detection function. We discuss different criteria for choosing the hazard function and consider in particular two different parametric families of hazard functions. Discrete and continuous hazard models are compared and the robustness of the discrete model is investigated. Finally, the methodology is applied to data from a survey for minke whales in the northeastern Atlantic.

The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site.

In Bacillus subtilis IS58 starved of glucose or exposed to heat shock, ethanol or salt stress, the sigmaB-dependent general stress protein GsiB is accumulated to a higher level than other general stress proteins. This high-level accumulation of GsiB can at least partially be attributed to the remarkably long half-life (approximately 20 min) of the gsiB mRNA. Analysis of different gsiB-lacZ fusions revealed that this stability is not determined by sequences at the 3' end of the transcript but rather by sequences upstream of the translational start codon. Site-directed mutagenesis established that a strong ribosome binding site was crucial for the increased stability of the gsiB mRNA. A comparison of the sequences upstream of the translational start codons of three general stress genes, gsiB, gspA and ctc, revealed a direct correlation between mRNA stability and the strength of their translational signals.

Health complaints and job stress in Norwegian physicians: the use of an overlapping questionnaire design.

An extensive research program has been undertaken in Norway on physician health, sickness, working conditions and quality of life. Data are collected from cross-sectional and longitudinal prospective and retrospective surveys, qualitative studies, and vital statistics. This paper presents findings on subjectively experienced health problems, emotional distress, experienced job stress and job satisfaction, based on an extensive cross-sectional postal questionnaire study in 1993. An overlapping questionnaire design was used to allow many relationships to be estimated without exhausting the recipients. 9266 active physicians were included, which comprises close to the total Norwegian physician work-force minus a representative sample of 2100, used for other studies. The primary questionnaire was returned by 6652 (71.8%), the great majority of which also returned three secondary questionnaires. The results indicate that health complaints were significantly more frequent in female physicians and decreased with age. Low job satisfaction, high job stress, and emotional distress were all found to be significant predictors of subjective health complaints, as measured by the Ursin Health Inventory.