Reconstitution and characterization of NtrC protein in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.     

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.     

Correlation between phylogenetic structure and function: examples from deep-sea Shewanella

The genus Shewanella is one of the typical deep-sea bacterial genera. Two isolated deep-sea Shewanella species, Shewanella benthica and Shewanella violacea, were found to be able to grow better under high hydrostatic pressure conditions than at atmospheric pressure. These species are not only piezophilic (barophilic), but also psychrophilic. Many psychrophilic and psychrotolerant Shewanella species have been isolated and characterized from cold environments, such as seawater in Antarctica or the North Sea. Some of these cold-adapted Shewanella were shown to be piezotolerant, meaning that growth occurs in a high-pressure habitat. In this review, we propose that two major sub-genus branches of the genus Shewanella should be recognized taxonomically, one group characterized as high-pressure cold-adapted species that produce substantial amounts of eicosapentaenoic acid, and the other group characterized as mesophilic pressure-sensitive species.     

Regulation of Cytochrome c- and Quinol Oxidases,and Piezotolerance of Their Activities in the Deep-Sea Piezophile Shewanella violacea DSS12 in Response to Growth Conditions

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O₂ concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O₂ concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O₂ concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.     

Diversity of piezophilic microorganisms in the closed ocean Japan Sea

Deep-sea sediment samples were collected at a depth of 3,064 m in the Japan Sea. Microorganisms in the sediment sample were cultivated under several pressure conditions, and the high-pressure adapted microbes were isolated. Two of the isolates exhibited piezophilic growth profiles. This is the first report to show the presence of piezophiles in the Japan Sea.     

The unique 16S rRNA genes of piezophiles reflect both phylogeny and adaptation

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few gamma-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.     

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.     

Pressure effects on in vivo microbial processes

Pressures between 10 and 100 MPa can exert powerful effects on the growth and viability of organisms. Here I describe the effects of elevated pressure in this range on mesophilic (atmospheric pressure adapted) and piezophilic (high-pressure adapted) microorganisms. Examination of pressure effects on mesophiles makes use of this unique physical parameter to aid in the characterization of fundamental cellular processes, while in the case of piezophiles it provides information on the essence of the adaptation of life to high-pressure environments, which comprise the bulk of our biosphere. Research is presented on the isolation of pressure-resistant mutants, high-pressure regulation of gene expression, the role of membrane lipids and proteins in determining growth ability at high pressure, pressure effects on DNA replication and topology as well as on cell division, and the role of extrinsic factors in modulating enzyme activity at high pressure.     

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR,UV/VIS and fluorescence spectroscopy study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.     

The rpsD gene, encoding ribosomal protein S4, is autogenously regulated in Bacillus subtilis.

Although the mechanisms for regulation of ribosomal protein gene expression have been established for gram-negative bacteria such as Escherichia coli, the regulation of these genes in gram-positive bacteria such as Bacillus subtilis has not yet been characterized. In this study, the B. subtilis rpsD gene, encoding ribosomal protein S4, was found to be subject to autogenous control. In E. coli, rpsD is located in the alpha operon, and S4 acts as the translational regulator for alpha operon expression, binding to a target site in the alpha operon mRNA. The target site for repression of B. subtilis rpsD by protein S4 was localized by deletion and oligonucleotide-directed mutagenesis to the leader region of the monocistronic rpsD gene. The B. subtilis rpsD leader exhibits little sequence homology to the E. coli alpha operon leader but may be able to form a pseudoknotlike structure similar to that found in E. coli.     

Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.     

Analysis of the Salmonella typhimurium proteome through environmental response toward infectious conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.     

Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned upstream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions.     

YycH regulates the activity of the essential YycFG two-component system in Bacillus subtilis

Of the numerous two-component signal transduction systems found in bacteria, only a very few have proven to be essential for cell viability. Among these is the YycF (response regulator)-YycG (histidine kinase) system, which is highly conserved in and specific to the low-G+C content gram-positive bacteria. Given the pathogenic nature of several members of this class of bacteria, the YycF-YycG system has been suggested as a prime antimicrobial target. In an attempt to identify genes involved in regulation of this two-component system, a transposon mutagenesis study was designed to identify suppressors of a temperature-sensitive YycF mutant in Bacillus subtilis. Suppressors could be identified, and the prime target was the yycH gene located adjacent to yycG and within the same operon. A lacZ reporter assay revealed that YycF-regulated gene expression was elevated in a yycH strain, whereas disruption of any of the three downstream genes within the operon, yycI, yycJ, and yycK, showed no such effect. The concentrations of both YycG and YycF, assayed immunologically, remained unchanged between the wild-type and the yycH strain as determined by immunoassay. Alkaline phosphatase fusion studies showed that YycH is located external to the cell membrane, suggesting that it acts in the regulation of the sensor domain of the YycG sensor histidine kinase. The yycH strain showed a characteristic cell wall defect consistent with the previously suggested notion that the YycF-YycG system is involved in regulating cell wall homeostasis and indicating that either up- or down-regulation of YycF activity affects this homeostatic mechanism.