Fifteen years of physiological proteo(geno)mics with (marine) environmental bacteria.

Environmental bacteria play a central role in the Earth's elemental cycles and represent a mostly untapped reservoir for novel metabolic capacities and biocatalysts. Over the last 15 years, the author's laboratory has focused on three major switches in the breakdown of organic carbon defined by the abundance and recalcitrance of the substrates: carbohydrates and amino acids by aerobic heterotrophs, fermentation end products by sulphate reducers and anaerobic degradation of aromatic compounds and hydrocarbons by denitrifiers and sulphate reducers. As these bacteria are novel isolates mostly not accessibly by molecular genetics, genomics combined with differential proteomics was early on applied to obtain molecular-functional insights into degradation pathways, catabolic and regulatory networks, as well as mechanisms and strategies for adapting to changing environmental conditions. This review provides some background on research motivations and briefly summarizes insights into studied model organisms, e.g. "Aromatoleum aromaticum" EbN1, Desulfobacula toluolica Tol2 and Phaeobacter inhibens DSM 17395.

The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments.

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.

Complete genome sequence of the marine planctomycete Pirellula sp. strain 1.

Pirellula sp. strain 1 ("Rhodopirellula baltica") is a marine representative of the globally distributed and environmentally important bacterial order Planctomycetales. Here we report the complete genome sequence of a member of this independent phylum. With 7.145 megabases, Pirellula sp. strain 1 has the largest circular bacterial genome sequenced so far. The presence of all genes required for heterolactic acid fermentation, key genes for the interconversion of C1 compounds, and 110 sulfatases were unexpected for this aerobic heterotrophic isolate. Although Pirellula sp. strain 1 has a proteinaceous cell wall, remnants of genes for peptidoglycan synthesis were found. Genes for lipid A biosynthesis and homologues to the flagellar L- and P-ring protein indicate a former Gram-negative type of cell wall. Phylogenetic analysis of all relevant markers clearly affiliates the Planctomycetales to the domain Bacteria as a distinct phylum, but a deepest branching is not supported by our analyses.

Anaerobic biodegradation of saturated and aromatic hydrocarbons.

Saturated and aromatic hydrocarbons are wide-spread in our environment. These compounds exhibit low chemical reactivity and for many decades were thought to undergo biodegradation only in the presence of free oxygen. During the past decade, however, an increasing number of microorganisms have been detected that degrade hydrocarbons under strictly anoxic conditions.

Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism.

A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism.

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes.

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.

Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria.

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., alpha-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the beta-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes.

Enzyme I(Ntr) from Escherichia coli. A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) phosphorylates sugars and regulates cellular metabolic processes using a phosphoryl transfer chain including the general energy coupling proteins, Enzyme I (EI) and HPr as well as the sugar-specific Enzyme II complexes. Analysis of the Escherichia coli genome has revealed the presence of 5 paralogues of EI and 5 paralogues of HPr, most of unknown function. The ptsP gene encodes an EI paralogue designated Enzyme I(nitrogen) (EI(Ntr)), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIA(Ntr), both implicated in the regulation of sigma(54) activity. The ptsP gene was polymerase chain reaction amplified from the E. coli chromosome and cloned into an overexpression vector allowing the overproduction and purification of EI(Ntr). EI(Ntr) was shown to phosphorylate NPr in vitro using either a [(32)P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EI(Ntr) phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EI(Ntr)/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EI(Ntr) has a pH optimum of 8.0, is dependent on Mg(2+), is stimulated by high ionic strength, and exhibits two K(m) values for NPr (2 and 10 microM) possibly because of negative cooperativity. The results suggest that E. coli possesses at least two distinct PTS phosphoryl transfer chains, EI(Ntr) --> NPr --> IIA(Ntr) and EI --> HPr --> IIA(sugar). Sequence comparisons allow prediction of residues likely to be important for specificity. This is the first report demonstrating specificity at the level of the energy coupling proteins of the PTS.

Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria.

Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide.

Anaerobic degradation of ethylbenzene and toluene in denitrifying strain EbN1 proceeds via independent substrate-induced pathways.

Denitrifying strain EbN1 utilizes either ethylbenzene or toluene as the sole source of organic carbon under strictly anoxic conditions. When cells were grown on ethylbenzene, 1-phenylethanol and acetophenone were detected in the culture supernatant. However, these two compounds were not observed when cells were grown on benzoate. Growth on ethylbenzene, 1-phenylethanol, or acetophenone strictly depended on the presence of CO2, whereas growth on benzoate did not. These results suggest that strain EbN1 degrades ethylbenzene via 1-phenylethanol and acetophenone as intermediates, and that acetophenone is subsequently carboxylated. In suspensions of benzoate-grown cells, induction was required for degradation of ethylbenzene, 1-phenylethanol, and acetophenone. Induction was also required for toluene-grown cells to gain activity to degrade ethylbenzene, and, conversely, for ethylbenzene-grown cells to degrade toluene. In accordance with our findings from these studies, two-dimensional gel electrophoretic analysis of extracts of cells grown on benzoate, acetophenone, ethylbenzene, or toluene showed that a number of substrate-specific proteins were induced in strain EbN1. Growth on toluene or ethylbenzene induced a distinct set of proteins. However, some of the induced proteins in ethylbenzene or acetophenone grown cells were identical. This agrees with the finding that acetophenone is an intermediate in the degradation of ethylbenzene.

Anaerobic utilization of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the beta-subclass of Proteobacteria.

Denitrifying bacteria were enriched from freshwater sediment with added nitrate as electron acceptor and crude oil as the only source of organic substrates. The enrichment cultures were used as laboratory model systems for studying the degradative potential of denitrifying bacteria with respect to crude oil constituents, and the phylogenetic affiliation of denitrifiers that are selectively enriched with crude oil. The enrichment culture exhibited two distinct growth phases. During the first phase, bacteria grew homogeneously in the aqueous phase, while various C1-C3 alkylbenzenes, but no alkanes, were utilized from the crude oil. During the second phase, bacteria also grew that formed aggregates, adhered to the crude oil layer and emulsified the oil, while utilization of n-alkanes (C5 to C12) from the crude oil was observed. During growth, several alkylbenzoates accumulated in the aqueous phase, which were presumably formed from alkylbenzenes. Application of a newly designed, fluorescently labelled 16S rRNA-targeted oligonucleotide probe specific for the Azoarcus/Thauera group within the beta-subclass of Proteobacteria revealed that the majority of the enriched denitrifiers affiliated with this phylogenetic group.

A novel protein kinase that controls carbon catabolite repression in bacteria.

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.

Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil.

A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m-xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m-isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 10(6) to 8 x 10(6) CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 10(5) CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.

Utilization of Alkylbenzenes during Anaerobic Growth of Pure Cultures of Denitrifying Bacteria on Crude Oil.

Four pure cultures of denitrifying bacteria, which had previously been isolated on defined alkylbenzenes, were capable of anaerobic growth with crude oil as the only source of organic substrates. Chemical analyses after growth revealed that the known growth substrates toluene, ethylbenzene, and m-xylene were selectively consumed from the oil. o-Xylene and p-xylene, which as pure compounds did not support growth, were consumed to a lesser extent.

Conversion studies with substrate analogues of toluene in a sulfate-reducing bacterium, strain Tol2.

Anaerobic toluene oxidation by the sulfate-reducing bacterium, strain Tol2 (proposed name Desulfobacula toluolica) was specifically inhibited by benzyl alcohol when added at concentrations around 500 microM. Benzyl alcohol added at lower, non-inhibitory concentrations (around 5 microM) was not oxidized by active cells pregrown on toluene, indicating that the alcohol is not a free intermediate of toluene metabolism in the sulfate reducer. Conversion of p-xylene in toluene-metabolizing cells to p-methylbenzoate as dead-end product suggests that the sulfate reducer, like denitrifiers, initiates toluene oxidation at the methyl group.

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria.

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluene, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO2 by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, stain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methyl-benzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.

Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria.

Many crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated. An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms. On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria, but their actual electron donors and carbon sources were unknown. On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ. Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions. A moderately thermophilic pure culture selectively utilizes n-alkanes in oil for sulphate reduction to sulphide. In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil. Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants.

Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium.

A toluene-degrading sulfate-reducing bacterium, strain Tol2, was isolated from marine sediment under strictly anoxic conditions. Toluene was toxic if applied directly to the medium at concentrations higher than 0.5 mM. To provide toluene continuously at a nontoxic concentration, it was supplied in an inert hydrophobic carrier phase. The isolate had oval, sometimes motile cells (1.2 to 1.4 by 1.2 to 2.0 microns). The doubling time was 27 h. Toluene was completely oxidized to CO2, as demonstrated by measurement of the degradation balance. The presence of carbon monoxide dehydrogenase and formate dehydrogenase indicated a terminal oxidation of acetyl coenzyme A via the CO dehydrogenase pathway. The use of hypothetical intermediates of toluene degradation was tested in growth experiments and adaptation studies with dense cell suspensions. Results do not support a degradation of toluene via one of the cresols or methylbenzoates, benzyl alcohol, or phenylacetate as free intermediate. Benzyl alcohol did not serve as growth substrate; moreover, it was a strong, specific inhibitor of toluene degradation, whereas benzoate utilization was not affected by benzyl alcohol. Sequencing of 16S rRNA revealed a relationship to the metabolically dissimilar genus Desulfobacter and on a deeper level to the genus Desulfobacterium. The new genus and species Desulfobacula toluolica is proposed.