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Urania basin in the deep Mediterranean Sea houses a lake that is >100 m deep, devoid of oxygen, 6 times more saline than seawater, and has very high levels of methane and particularly sulfide (up to 16 mM), making it among the most sulfidic water bodies on Earth. Along the depth profile there are 2 chemoclines, a steep one with the overlying oxic seawater, and another between anoxic brines of different density, where gradients of salinity, electron donors and acceptors occur. To identify and differentiate the microbes and processes contributing to the turnover of organic matter and sulfide along the water column, these chemoclines were sampled at a high resolution. Bacterial cell numbers increased up to a hundredfold in the chemoclines as a consequence of elevated nutrient availability, with higher numbers in the upper interface where redox gradient was steeper. Bacterial and archaeal communities, analyzed by DNA fingerprinting, 16S rRNA gene libraries, activity measurements, and cultivation, were highly stratified and metabolically more active along the chemoclines compared with seawater or the uniformly hypersaline brines. Detailed analysis of 16S rRNA gene sequences revealed that in both chemoclines [delta]- and [epsilon]-Proteobacteria, predominantly sulfate reducers and sulfur oxidizers, respectively, were the dominant bacteria. In the deepest layers of the basin MSBL1, putatively responsible for methanogenesis, dominated among archaea. The data suggest that the complex microbial community is adapted to the basin's extreme chemistry, and the elevated biomass is driven largely by sulfur cycling and methanogenesis. deep anoxic hypersaline lake | element cycling | geosphere--biosphere interaction | Mediterranean Sea | microbial diversity

The rate, timing, and quality of resource supply exert strong controls on a wide range of ecological processes. In particular, resource-mediated changes in microbial activity have the potential to alter ecosystem processes, including the production and respiration of organic matter. In this study, we used field experiments and simulation modeling to explore how aquatic heterotrophic bacteria respond to variation in resource quality (low vs. high) and resource schedule (pulse vs. press). Field experiments revealed that one-time pulse additions of resources in the form of dissolved organic carbon (DOC) caused short-lived ([less than or equal to] 48 h) peaks in bacterial productivity (BP), which translated into large differences across treatments: cumulative BP was twice as high in the pulse vs. press treatment under low resource quality, and five times as high under high resource quality. To gain a more mechanistic understanding of microbial productivity in variable resource environments, we constructed a mathematical model to explore the attributes of bacterial physiology and DOC supply that might explain the patterns observed in our field experiments. Model results suggest that the mobilization rate of refractory to labile carbon, an index of resource quality, was critical in determining cumulative differences in BP between pulse and press resource environments ([BP.sub.Pu:Pr] ratios). Moreover, [BP.sub.Pu:Pr] ratios were substantially larger when our model allowed for realistic changes in bacterial growth efficiency as a function of bacterial carbon consumption. Together, our field and modeling results imply that resource schedule is important in determining the flow of material and energy from microbes to higher trophic levels in aquatic food webs, and that the effects of resource quality are conditional upon resource schedule. An improved understanding of the effects of resource variability on microorganisms is therefore critical for predicting potential changes in ecosystem functioning in response to environmental change, such as altered DOC fluxes from terrestrial to aquatic ecosystems. Key words: bacteria; C[O.sub.2]; dissolved organic carbon; episodic; microbial physiology; subsidies; temporal variability; terrestrial-aquatic linkages.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifiuoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the 'TFA inactivation protocol' was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDITOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactiva tion of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Understanding the mechanisms and processes that generate biological diversity is a fundamental problem in evolution and ecology. In the past decade, the theory of evolutionary branching and adaptive diversification has provided new perspectives for understanding the evolution of diversity caused by ecological interactions. In models of adaptive diversification, the fitness landscapes change dynamically, so that the likelihood of diversification into different phenotypic clusters increases over time. In contrast, in models with static fitness landscapes, the likelihood of diversification decreases as populations climb fitness peaks, because crossing maladaptive fitness valleys becomes increasingly difficult. We used experimental evolution in bacteria to test how the likelihood of diversification changes over time in a bacterial lineage that has diversified in sympatry from a single ancestral strain. By analyzing the 'fossil' record of this lineage, and restarting the lineage from different time points in the evolutionary past, we demonstrate that: (i) the lineage has initially undergone a phase of directional adaptation to the competitive environment, and (ii) during this phase, the likelihood of diversification increases significantly over time. These results suggest evolutionary branching caused by frequency-dependent competition as the main mechanism of diversification in our experimental populations. Escherichia coli | evolutionary branching | experimental evolution | frequency dependence

High-molecular-weight dissolved organic matter (HMW-DOM, > 1,000 Daltons) is actively involved in the global biogeochemical cycling of many elements, but its carbon sources and detailed formation pathways are still not well understood. In this study, we measured bulk stable carbon and nitrogen isotopic ratios, lipid composition, and compound-specific carbon isotopic ratios of HMW-DOM samples collected from four U.S. estuaries (Boston Harbor/Massachusetts Bay, Delaware/Chesapeake Bay, San Diego Bay, and San Francisco Bay). Analytical results show (1) a fraction of HMW-DOM (lipid associated) in estuarine and coastal waters is derived from bacteria and phytoplankton; (2) this fraction of HMW-DOM is formed by various release processes of bacterial membrane components and bacterial reworking of phytoplankton-derived material; (3) this fraction of HMW-DOM is generally present in all samples from different coastal systems despite variable organic matter inputs and environmental conditions, suggesting an important bacterial role in HMW-DOM formation.

Most studies of spring bloom succession in Norwegian waters have employed light microscopy and accounted for species composition of phyto- and zooplankton. Flow cytometry and molecular tools enable us to extend such investigations to include smaller organisms like bacterio- and virioplankton. Here, we describe succession and diversity of algae, bacteria, and viruses in relation to environmental changes from 15 February to 27 April. The spring succession started with an increase in autotrophic picoeukaryotes and Synechococcus sp. The diatoms bloomed around the middle of March and caused nutrient depletion in the upper part of the water column. Upwelling in the beginning of April gave rise to a second bloom, consisting of diatoms and Phaeocystis pouchetii. Numerically, autotrophic picoeukaryotes and Synechococcus sp. dominated the periods between and after these two major blooms. Heterotrophic bacterial abundance increased throughout the experimental period and reached peak values during and after phytoplankton blooms. These bacteria were succeeded by viruses having low DNA fluorescence, whereas viruses with medium DNA fluorescence bloomed during or after blooms of autotrophic picoeukaryotes. High-DNA fluorescence viruses reached maximum concentrations during and after the diatom and Phaeocystis blooms. The diversity of the bacterial community remained relatively stable, whereas viral diversity varied more and increased after major phytoplankton blooms. Our investigation thus demonstrates how virioplankton are important elements of the total microbial diversity and how they are intimately linked to the rest of the microbial community and possibly act as an internal driving force in spring bloom successions.

We investigated bacterial responses to variations in dissolved organic carbon (DOC) and nutrient availability by a comparative analysis of bacterial metabolism in lakes ranging from oligotrophic to eutrophic. Bacterial growth, respiration, and growth efficiency were quantified in lake water dilution cultures performed in 20 lakes located in eastern Quebec, Canada, which varied with respect to both DOC and nutrient concentrations. Intrinsic growth rates of the bacteria ranged from 0.1 to 1.4 [d.sup.-1], bacterial cell-specific respiration rates ranged from 0.4 to 7.2 fg C [cell.sup.-1] [h.sup.-1], and growth efficiencies ranged from 6.7% to 51.6%. These variations were unrelated to bulk DOC concentrations. Instead, growth rate and efficiency were positively related to total phosphorus concentrations. Specific respiration rate, on the other hand, decreased with increasing phosphorus concentrations, and the magnitude of respiration, on a per-cell basis, strongly influenced observed growth efficiencies. In a series of substrate enrichment experiments, additions of glucose alone failed to stimulate a response in growth rate, mean cell biovolume, or the potential biomass yield in dilution cultures, but all responded positively to phosphorus additions. Our results show that bacterial metabolism and the fate of DOC input to lake microbial communities are strongly dependent on phosphorus availability, rather than total carbon availability. Extreme oligotrophy appears to place high respiratory demands on the bacterioplankton, resulting in very low bacterial growth efficiencies and consequently greater DOC flow to C[O.sub.2] than to biomass available for transfer to higher trophic levels.

The influence of inlet bacteria on the assemblage composition of bacterioplankton was investigated in two Swedish forest lakes of different hydraulic retention time. Assemblage composition of the bacteria in lakes and running waters was determined by denaturing gradient gel electrophoresis (DGGE) and sequencing of polymerase chain reaction (PCR)-amplified 16S rDNA. The amount of bacterial cells imported via the inlets in relation to bacterioplankton cells produced in the epilimnion of the lakes was also determined. In the lake with short retention time (theoretical hydraulic retention time of 0.3 yr) the lake bacterioplankton assemblage largely resembled the riverine assemblages, although the extent of similarity varied among inlets, depending on water flow. In the lake with long retention time (theoretical hydraulic retention time of 10 yr) the bacterioplankton assemblage in the lake had low similarity to the inlet assemblages. The degree of similarity between inlets and lakes was well correlated to the amount of imported cells. Thus, our data suggest that import of inlet bacteria could have a large effect on the composition of lake bacterioplankton assemblages and that hydrological factors determined the magnitude of this effect. Since short hydraulic retention times are very common in lakes in the boreal zone, input of allochthonous bacteria can be one major factor influencing bacterioplankton assemblage composition in boreal lakes.

An autostimulatory substance produced by Escherichia coli has been characterized. The autostimulatory effect of the E. coli supernatants is independent of the requirement for carbon dioxide.

Trehalose, a nonreducing disaccharide found at nigh concentrations in the filamentous fungus Aspergillus nidulans conidia and rapidly degraded with induction of conidial germination, has been found to be required for acquisition of tolerance to various stresses in the fungus. It appears that trehalose 6-phosphate is a physiological inhibitor of hexokinase, but that the control is not essntial for proper glycolytic flux in a. nidulans. Also tpsA transcription is not induced in resonse to heat shock or in conidiation.

Genes with a role in the activation of the Bacillus thuringiensis immune inhibitor A (inhA) metalloprotease gene at the onset of sporulation have been identified. Study has shown that the major regulator of inhA expression is AbrB. It acts as a repressor of inhA transcription.

An enantioselective amidase from Agrobacterium tumefaciens strain d3 has been characterized genetically and biochemically. An effort was made to establish a structure-function relationship for the conversion of various amides carrying different substituents adjacent to the amide group with respect to the reaction rate and the enantioselectivity of the amidase.

Characterization and separation of individual mycolic acids has been undertaken in 24 representative mycobacteria. The stereochemistry of the double bonds and cyclopropane rings and the chain lengths of each of the separated component mycolates was assayed.

Cell-wall polysaccharides from Nectria cinnabarina have been compared with those from the group of Vectria with Sesquicillium anamorphs. These polysaccharides are alkali-extractable and water-soluble.

A Bacillus amyloloquefaciens chitin-binding (ChbB) protein, which has homologues in related strains, has been found to bind alpha- and beta-chitin. ChbB is secreted by B amyloliquefaciens ALDO 2718. It cross-reacts with anti-CHB1 antibodies.

YkrB has been found to be the main peptide deformylase in Bacillus subtilis and B subtilis has two functional peptide deformylases, unlike all other eubacteria thusfar studied. For the first time, biochemical and shysiological roles of two functional Def proteins derived from one organism are compared. . B subtilis has a deformylase-like gene called ykrB. Its product is much like the class II deformylase from B. stearothermophilus.

Escherichia coli OsmC, an osmotically inducible protein, the function of which is not known, and Xanthomonas campestris Ohr, a protein that has a role in organic peroxide protection, are related. They cluster in two related subfamilies of proteins widely distributed in Gram-negative/positive bacteria. Pseudomonas aeruginosa and Deinococcus radiodurans, each having one member of each subfamily, have been studied with the finding that bacterial OsmC and Ohr paralogues define two protein families having distinct patterns of expression and functions.

Corynebacterium glutamicum and expression control and specificity of its basic amino acid exporter LysE are discussed. Lys E exports L-lysine and L-arginine at comparable rates. L-histidine and L-citrulline act as coinducers of lysE expression but are not exported by LysE.

Pleurotus sajor-caju and differential regulation of laccase gene expression in this edible mushroom have been studied. A high degree of homology was seen in the genes vs other basidiomycete laccases at the amino acid level.

A halobacterial bgaH reporter gene has been used to analyze regulation of gene expression in halophilic archaea. Genes encoding gas vesicles were used to study regulation. Haloferax volcanii transformants were produced containing fusions between the various gvpA promoter regions and the bgaH reading frame.

Closely related Prochlorococcus genotypes have been shown to have remarkably different depth distsributions in the Red Sea and the North Atlantic. An in situ hybridization method was used for identification of marine cyanobacteria using horseradish-peroxidase-labelled 16S rRNA targeted oligonucleotide probes in combination with tyramid signal amplification (TSA).

Repeated ruminal dosing of Ruminococcus spp. has been found not to result in persistence in studies related to fiber digestibility and whether it is the cellulolytic bacteria or the nature of plant cell walls that are the limiting factor. Changes in other microbial populations can be measured with unique quantitative 16S-rRna-based probes. Higher level probes were used to explore the effects of dosing on Fibrobacter, Ruminococcus, and eukaryotes.

Green fluorescent protein (GFP) fluorescence in Streptococcus gordonii was assessed under low-oxygen concentration and low-pH conditions. GFP, it was found, can be used in studies of oxygen-tolerant anaerobic bacteria. Densely packed, flow cell-grown biofilms of S. gordonii do not develop oxygen gradients that inhibit GFP fluorescence development. The transient nature of GFP fluorescence in acid-producing bacteria can often be overcome in flowcells.

The Mesorhizobium sp. strain R7A bio operon on its acquired symbiosis island of has been found to include a novel gene that is part of the pimeloyl-coA synthesis. The reason the vitamin biosynthetic loci are on an acquired genetic element not in nonsymbiotic mesorhizobia is not yet known. The symbiosis island is a 500-kb chromosomal genetic elements that when transferred converts nonsymbiotic mesorhizobia to symbionts that can nodulate and fix nitrogen with Lotus corniculatus. A 13.2-kb region of the island that changed the nonsymbionts to nicotinate and biotin prototrophy has been characterized.

Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Streptomyces aureofaciens has been studied and found to require GapR. The gapR gene was constitutively expressed from one promoter during differentiation. By integrative transformation using double crossover, a stable null mutant of the gapR gene was obtained. The mutation affected growth little and differentiation not at all. Transcription of the GAPDH-encoding gap gene was substantially reduced in the S. aureofaciens (Delta)gapR null mutant, no matter what the carbon source. GapR is a member of the AraC/XyIS family of transcriptional activators.

Neisserial TonB-dependent outer-membrane proteins are discussed. A study used detection, regulation and distribution of three putative candidates identified from the genome sequences. One of the candidates is a potential vaccine candidate. Expression of TdfH is not affected by iron availability. .

It has been shown that glnD is an essential gene in Azotobacter vinelandii. Lethality of glnD null mutations in A. vinelandii has been found to be suppressible by prevention of glutamine synthetase adenylylation.

A Staphylococcus aureus Zn(super.2+)-responsive regulatory element is discussed. The putative operon, zur, and components of an ABC-type transporter have been characterized, and it appears zur has no role in pathogenicity in a lesion model of S. aureus infection..

Strong expression signals of Mycobacterium tuberculosis have been isolated using the natural fluorescence of the Aequoria victoria green fluorescent protein. Two of the promoters corresponded to proteins thought to be involved in magnesium and calcium utilization, very important for cell physiology and thus controlled by strong transctiption signals. The strength of the expression signals could be used for overexpression of foreign genes in mycobacteria to help with vaccine vector development and protein purification. Fluorescence-activated cell sorting (FACS) has been shown to offer a sensitive, efficient means to measure and select strong mycobacterial expression signals.

Three tetracycline resistance elements have been found to be closely related. Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis, and the CW459tet(M) element from Clostridium perfringens have been compared. They were found to have similar conjugation regions despite different excision and insertion modules.

Cyclic heptapeptide microcystin biosynthesis has been found to require the glutamate racemase gene in the first reported research of the racemase in prokaryotic nonribosomal peptide synthetase (NRPS). The role of mcyF in microcystin synthesis has been studied using gene-disruption and complementation analyses.

Identity elements in tRNA-mediated transcription antitermination have been studied with attention to implications of tRNA D- and T-arms in mRNA recognition. The mRNA-tRNA interaction involves parts of the tRNA in addition to the anticodon and the acceptor in an earlier model. The additional interactions may contribute to the recognition of a specific tRNA, and thus to the specificity and efficacy of the regulatory response.

The histidine kinase (HK) family in bacteria and archaea has been studied using genomic analysis. Archeal genomes had either a specific HK type or no HKs at all, but several bacterial genomes had all five HK types. Findings suggest different HK types originated in bacteria. It appears that specific HK types went into archaea by horizontal gene transfer.

A physical chromosome map has been made of the European stone fruit yellows (ESFY) phytoplasma strain GSFY1 as a first step toward a detailed genetic characterization of the ESFY phytoplasma. ESFY phytoplasma is an important prokaryotic pathogen that infects most kinds of stone fruits in Europe. The map, made using PFGE-purified genomic DNA from diseased tobacco and tomato plants, was not identical to that of the closely related apple proliferation phytoplasma, but was similar.

Streptococcus thermophilus lacZ has been used as a versatile reporter gene for Candida albicans. It has been shown that beta-galactosidase activity can readily be detected from several C. albicans promoters, given the right growth conditions.

Fibre fluorescence in situ hybridization (fibre-FISH) has been applied to an ascomycete. The goal was visualization of the Cochliobolus heterostrophus rRNA gene cluster. C. heterostrophus was found to have 90+ copies of the repeat unit in its rDNA. The stretching degree was similar to the value based on the Watson-Crick model. Fibre-FISH is not known to have been used in filamentous fungi.

Isolation of novel avirulence genes from Pseudomonas syringae pv. pisi is discussed. Highly conserved sequences were found to flank avirulence genes, meaning implications for the horizontal transfer of avirulence and virulence genes exist.

The outer membranes (OMs) of the pathogenic spirochaetes Borrelia burgdorferi and Treponema pallidum have been studied using insertion of fluorescent fatty acid (FA) probes. Support was found for the prediction that lack of lipopolysaccharide makes T. pallidum and B. burgdorferi OMs decidedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts. It seems the two spirochaetes may obtain long-chain FAs, nutrients they cannot synthesize, through direct OM permeation.

Overexpression of wild-type and mutant regulatory subunits in Saccharomyces cerevisiae has been used for evaluation of in vivo activation of protein kinase A under non-dissociable conditions. This overexpression is a new approach. Biochemical and genetic results support the hypothesis that within the cell it is possible to catalytically active, cAMP-bound, undissociated protein kinase (PKA) holoenzyme.

Eubacterial arylamine N-acetyltransferases (NATs) are discussed based on identification and comparison of 18 members of the protein family. A study was undertaken with the goal of determining how widespread NAT is in the prokaryotes. Also of interest was to what extent conserved residues are maintained. Conserved active site cysteine, histidine, and aspartate residues were found.

A microbial aquaglyceroporin from Lactococcus lactis has been functionally characterized. Aquaglyceroporins are one class of major intrinsic proteins (MIPs), which make up a widespread membrane channel family required for osmotic cell equilibrium. Bacterial MIP genes are in archaea and in eubacteria, both Gram-positive and Gram-negative. The MIP is permeable to glycerol and to water.

Pseudomonas aeruginosa mutations in rhll and lasl quorum sensing systems bring milder chronic lung infection. In vivo pathogenic effects of the wild-type P. aeruginosa PAO1 and its double mutation, PAO1 lasl rhll, in which the signal-generating parts of the quorum sensing systems are defective, have been compared. The goal was to find out about importance of quorum sensing in chronic P. aeruginosa lung infection.

Listeria monocytogenes isolates from animal and human listeriosis cases have been subjected to comparative genetic characterization using automated ribotyping and PCR-restriction fragment length polymorphism (RFLP) typing of the virulence genes hly and actA. Results may show host specificities or unique pathogenic potentials. Ribotyping, especially with PCR-RFLP of hyl and actA virulence genes, is an effective way to differentiate/characterize L. monocytogenes isolates.

Comamonas testosteroni lipid A and its endotoxic properties are discussed. C. testosteroni is an aerobic, Gram-negative rod that grows on testosterone, and its lipid A is very homogeneous vs other naturally produced lipid As. It appears that the composition of 3- hydroxydecanoic acid is not responsible for the low endotoxicity of lipd A seen in the genus Rhodopseudomonas.

Host specificity and geographical structure in bacteria are discussed in this minireview article with consideration of implications for tracing coliform-contamination sources. It may be that PCR-based detection of virulence factors would give better indication of the potential health significance of coliforms in the environment. It might be better for tracing sources of contamination in the environment. Topics include E. coli. genetic structure and virulence factors, sampling considerations, temporal variation in populations, whether Escherichia coli is the right coliform to look at, and primary vs secondary habitats.

The promoter of the Paracoccus denitrificans nitric oxide (NO) reductase operon (norCBQDEF) has been studied using primer extension and deletion analysis. Two transcription-start sites were seen, the major one active only in cultures grown anaerobically, with nitrate, and is 45.5 bp downstream of the putative NNR-binding site, so this is a class II factor-dependent promoter. NNR is the sole regulatory protein with a significant role in regulating expression of the NO reductase. The NNR-binding site has been confirmed.

The Bacillus subtilis gene ftsY is expressed only at t(sub.8) during sporulation and is from a promoter controlled by sigma(super.K) and GerE and immediately upstream of ftsY and inside the smc gene. B. subtilis FtsY (Srb) is a homolog of the alpha subunit of the receptor for the mammalian signal-recognition particle and is required for vegetative cell growth and protein secretion. To look at the role of FtsY in sporulation, the B. subtilis strain ISR39 ws established. It is an ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative stage.

Phylogeny of the genus Pseudomonas is discussed with attention to intrageneric structure derived based on the nucleotide sequences of gyrB and rpoD genes and phylogenetic analysis. Classification criteria for Pseudomonas as a genus have been revised. Two major clusters, intrageneric clusterI and intrageneric cluster II, have emerged from the study.

Production of phytotoxin coronatine (COR) in defined hrp/hrc mutants of Pseudomonas syringae pv tomato DC3000 has been analyzed. The goal was to study the coordinated regulation, if any existed, of different systems necessary for virulence and pathogenicity. Results show it is likely that the hrp/hrc secretion system is not necessary for COR production. Mutations in the system however may have regulatory effects on production of virulence factors of the COR type