Showing papers in "Journal of Bacteriology in 2003"
TL;DR: It is suggested that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression, and acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes during P. aeruginosa growth in culture.
Abstract: There are two interrelated acyl-homoserine lactone quorum-sensing-signaling systems in Pseudomonas aeruginosa. These systems, the LasR-LasI system and the RhlR-RhlI system, are global regulators of gene expression. We performed a transcriptome analysis to identify quorum-sensing-controlled genes and to better understand quorum-sensing control of P. aeruginosa gene expression. We compared gene expression in a LasI-RhlI signal mutant grown with added signals to gene expression without added signals, and we compared a LasR-RhlR signal receptor mutant to its parent. In all, we identified 315 quorum-induced and 38 quorum-repressed genes, representing about 6% of the P. aeruginosa genome. The quorum-repressed genes were activated in the stationary phase in quorum-sensing mutants but were not activated in the parent strain. The analysis of quorum-induced genes suggests that the signal specificities are on a continuum and that the timing of gene expression is on a continuum (some genes are induced early in growth, most genes are induced at the transition from the logarithmic phase to the stationary phase, and some genes are induced during the stationary phase). In general, timing was not related to signal concentration. We suggest that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression. Acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes during P. aeruginosa growth in culture.
1,082 citations
TL;DR: The profound influence of the physics of the diffusion process on the chemistry and biology of the biofilm mode of growth is discussed.
Abstract: Much of what makes life in a microbial biofilm different from life in a free aqueous suspension can be explained by invoking the phenomenon of diffusion. This article discusses the profound influence of the physics of the diffusion process on the chemistry and biology of the biofilm mode of growth.
1,032 citations
TL;DR: Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated, and a large number of additional QS-promoted genes were identified.
Abstract: Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P ≤ 0.05) in response to the exogenous autoinducers and were classified as QS regulated. A total of 244 genes were identified as being QS regulated at the mid-logarithmic phase, and 450 genes were identified as being QS regulated at the early stationary phase. Most of the previously reported QS-promoted genes were confirmed, and a large number of additional QS-promoted genes were identified. Importantly, 222 genes were identified as being QS repressed. Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated.
908 citations
TL;DR: A genetic footprinting technique is used for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media to identify 620 genes as essential and 3,126 genes as dispensable for growth under these conditions.
Abstract: Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.
762 citations
TL;DR: It is proposed that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities, and evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces is provided.
Abstract: In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.
752 citations
TL;DR: The first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing, show that the recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.
Abstract: Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.
741 citations
TL;DR: Examination of the sequence changes at MLST loci during clonal diversification shows that point mutations give rise to new alleles at least 15-fold more frequently than does recombination, which suggests that homologous recombination does contribute toward the evolution of this species over the long term.
Abstract: Staphylococcus aureus is an important human pathogen and represents a growing public health burden owing to the emergence and spread of antibiotic-resistant clones, particularly within the hospital environment. Despite this, basic questions about the evolution and population biology of the species, particularly with regard to the extent and impact of homologous recombination, remain unanswered. We address these issues through an analysis of sequence data obtained from the characterization by multilocus sequence typing (MLST) of 334 isolates of S. aureus, recovered from a well-defined population, over a limited time span. We find no significant differences in the distribution of multilocus genotypes between strains isolated from carriers and those from patients with invasive disease; there is, therefore, no evidence from MLST data, which index variation within the stable “core” genome, for the existence of hypervirulent clones of this pathogen. Examination of the sequence changes at MLST loci during clonal diversification shows that point mutations give rise to new alleles at least 15-fold more frequently than does recombination. This contrasts with the naturally transformable species Neisseria meningitidis and Streptococcus pneumoniae, in which alleles change between 5- and 10-fold more frequently by recombination than by mutation. However, phylogenetic analysis suggests that homologous recombination does contribute toward the evolution of this species over the long term. Finally, we note a striking excess of nonsynonymous substitutions in comparisons between isolates belonging to the same clonal complex compared to isolates belonging to different clonal complexes, suggesting that the removal of deleterious mutations by purifying selection may be relatively slow.
637 citations
TL;DR: It is proposed that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells in biofilms of P. aeruginosa.
Abstract: Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.
589 citations
TL;DR: This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria as well as predicted insertion sequence elements in eight different families.
Abstract: Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with ∼47% transcribed from one strand and ∼53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO 2 and NH 3 assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.
514 citations
TL;DR: In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF, providing additional evidence for the hypothesis that Cu(I) is directly transported from the periplasms across the outer membrane by the Cus complex.
Abstract: The cus determinant of Escherichia coli encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux. CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance. Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance. Substitutions for several other methionine residues of this protein did not have any effect. The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein. CusF bound one copper per polypeptide. The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm. Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis. CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay. In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF. These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex.
483 citations
TL;DR: This study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state, and homeostatic control of cysteine levels is important in conferring resistance to oxidants.
Abstract: Escherichia coli is generally resistant to H2O2, with >75% of cells surviving a 3-min challenge with 2.5 mM H2O2. However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H2O2, with <1% surviving the challenge. Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay. Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H2O2 into a hydroxyl radical, the direct effector of DNA damage. The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold. In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed. This action permits free iron to redox cycle rapidly in the presence of H2O2, thereby augmenting the rate at which hydroxyl radicals are formed. During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage. Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants. More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state.
TL;DR: The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined and confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria.
Abstract: The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined. Whole-genome comparative analysis with other available complete genome sequences confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria. Within the CFB phyla, the genomes most similar to that of P. gingivalis are those of Bacteroides thetaiotaomicron and B. fragilis. Outside of the CFB phyla the most similar genome to P. gingivalis is that of Chlorobium tepidum, supporting the previous phylogenetic studies that indicated that the Chlorobia and CFB phyla are related, albeit distantly. Genome analysis of strain W83 reveals a range of pathways and virulence determinants that relate to the novel biology of this oral pathogen. Among these determinants are at least six putative hemagglutinin-like genes and 36 previously unidentified peptidases. Genome analysis also reveals that P. gingivalis can metabolize a range of amino acids and generate a number of metabolic end products that are toxic to the human host or human gingival tissue and contribute to the development of periodontal disease.
TL;DR: Findings suggest that beta-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers, strongly supporting the hypothesis of the unique origin of common nod genes.
Abstract: Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948–950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial-proteobacteria (-rhizobia) are very similar to those of rhizobia from the-subclass (-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered-rhizobial sequences in two nodA lineages intertwined with-rhizobial sequences. On the other hand, the-rhizobia were grouped with free-living nitrogen-fixing-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers
TL;DR: High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure.
Abstract: High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space. It is possible that the protoplast has some capacity to float freely within the periplasm.
TL;DR: It is concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.
Abstract: Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus that colonizes the human oral cavity (20). A. actinomycetemcomitans has been implicated as the causative agent of localized juvenile periodontitis, a severe and rapid form of periodontal disease that affects adolescents (38). A. actinomycetemcomitans can also enter the submucosa and cause infective endocarditis and other nonoral infections (15). When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite (5, 6, 9, 12-14, 16, 18, 20, 28). Nearly all of the cells grow attached to the surface, while the broth remains clear and is often sterile (5). The dense biofilm that forms on the surface is resistant to removal by agents such as detergents, proteases, heat, sonication, and vortex agitation and can be removed only by mechanical scraping or by treatment with the carbohydrate-modifying reagent periodic acid (5). A. actinomycetemcomitans biofilm cells exhibit increased resistance to antimicrobial agents compared to the resistance exhibited by cells grown in planktonic form (4). Tight adherence has been shown to play an important role in the ability of A. actinomycetemcomitans to colonize the mouths of rats (7) and probably plays an equally important role in its ability to colonize humans. Tight adherence to surfaces is dependent on the presence of long, bundled pili (fimbriae) that form on the surface of the cell (12, 28). Mutations in flp-1, which encodes the major pilin protein subunit, result in cells that fail to produce fimbriae or adhere to surfaces (14).
Kaplan and Fine showed that biofilm colonies of A. actinomycetemcomitans release cells into liquid medium and that these cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread (16). Kaplan et al. isolated three A. actinomycetemcomitans transposon insertion mutants that formed biofilm colonies which were tightly adherent to surfaces but which failed to release cells into the medium or spread over the surface (18). All three of the transposon insertions mapped to genes required for the synthesis of the O-polysaccharide (O-PS) component of lipopolysaccharide. Microscopic analysis of the O-PS mutants indicated that they lacked a layer of nonaggregated cells that was present inside biofilm colonies of the wild-type parental strain. These findings led to the hypothesis that A. actinomycetemcomitans biofilm cell detachment occurs by means of a novel mechanism that involves the release of cells from inside the biofilm colony (18).
In this report we describe a fourth A. actinomycetemcomitans transposon insertion mutant that is deficient in biofilm cell detachment and biofilm dispersal. The transposon in this strain inserted into a novel gene, designated dspB, which encodes a protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. By using DspB protein purified from an overexpressing strain of Escherichia coli, we obtained evidence that dspB encodes a soluble N-acetylglucosaminidase that causes the detachment of cells from A. actinomycetemcomitans biofilm colonies.
TL;DR: The 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2 is presented, a human-specific pathogen causing typhoid fever, and a half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed.
Abstract: We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.
TL;DR: A protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility, is established and the data suggest an evolution of mcy clusters from genes for nodularin (a related pentapeptide) biosynthesis.
Abstract: Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.
TL;DR: The results suggest that Streptococcus gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism, and LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. g Gordonii.
Abstract: Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-β-d-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.
TL;DR: The data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes.
Abstract: While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, the σB regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate σB-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant σB-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, the σB-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that σB contributes to L. monocytogenes gene expression during infection.
TL;DR: The construction of in silico models represents a way to interpret data from the recent flood of genomic, transcriptomic, and other high-throughput data in a systemic fashion.
Abstract: The recent flood of genomic ([13][1]), transcriptomic ([29][2]), and other high-throughput data ([21][3], [33][4], [45][5]) makes the need to interpret this information in a systemic fashion increasingly pressing. The construction of in silico models represents a way to interpret these data and
TL;DR: Results indicate that OmcB, but not OmcC, has a major role in electrons transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe( III) reduction in this organism.
Abstract: Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.
TL;DR: It is demonstrated that phiBT1 integrates into a different attachment site than phiC31, which lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein.
Abstract: Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.
TL;DR: Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays.
Abstract: Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.
TL;DR: 100 mutants are tested, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR- regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS, which provide a wealth of new information concerning many of these systems.
Abstract: Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.
TL;DR: The σ54 subunit is a unique sigma subunit required for promoter recognition and initiation of transcription by the bacterial RNAP that resembles the enhancer-binding proteins (EBPs) found in eukaryotic systems, and for this reason such activators are known as bacterial EBPs.
Abstract: Transcription is the key control point for regulation of numerous cellular activities. Bacteria regulate levels of gene expression by using transcription factors that modulate the recruitment of RNA polymerase (RNAP) to promoter elements in the DNA. Many bacteria also control gene expression by using a second class of transcription factor that uses energy from nucleotide hydrolysis to actively promote transcription initiation.
The sigma (σ) subunit is required for promoter recognition and initiation of transcription by the bacterial RNAP. Typically a bacterial cell may contain several alternative σ subunits with differing sequence specificities that direct the RNAP holoenzyme to different sets of promoters. The σ54 subunit (also known as RpoN, NtrA, and σN) is unique (42) in that it shares no detectable homology with any of the other known sigma factors (e.g., σ70 and σ28). σ54-RNAP binds to specific promoter sites, with the consensus DNA sequence YTGGCACGrNNNTTGCW (6), to form a transcriptionally inactive closed complex consisting of holoenzyme bound to double-stranded DNA. In contrast to σ70-RNAP bound at its cognate promoter sites, σ54-RNAP is unable to spontaneously isomerize from a closed complex to a transcriptionally competent open complex (11, 72). To proceed with initiation of transcription, the closed complex must participate in an interaction with a transcriptional activator, involving nucleotide hydrolysis. This transcriptional activator is usually bound at least 100 bp upstream of the promoter site, and DNA looping is required for the activator to contact the closed complex and catalyze formation of the open promoter complex. In this respect the activator resembles the enhancer-binding proteins (EBPs) found in eukaryotic systems, and for this reason such activators are known as bacterial EBPs.
From a protein structural point of view, EBPs share in common a σ54 interaction module (Pfam accession number PF00158) but typically have at least one additional domain (Fig. (Fig.1).1). In nearly all of those investigated so far, there is a DNA-binding domain containing a helix-turn-helix sequence motif, enabling the protein to bind to specific DNA enhancer elements upstream of σ54-dependent promoters (44, 47, 52, 56, 72). One exception to this scenario was recently reported (30), where Pseudomonas aeruginosa FleQ can activate transcription while bound in the downstream vicinity of the promoter.
FIG. 1.
Major domain architectures of bacterial EBPs. Examples of each of the known domain organizations found in bacterial EBPs are given. Sequences are identified by SwissProt/trEMBL accession numbers, except for XAC3643, TTE0180, and {"type":"entrez-protein","attrs":{"text":"CPE23358","term_id":"896862659","term_text":"CPE23358"}} ...
Although the physiological advantages conferred by the σ54-EBP mode of transcription are not yet clear, activation of σ54-dependent transcription is highly regulated by environmental cues through regulatory modules in the EBPs and in some cases by interactions with other regulatory proteins. Sensory modules in EBPs include CheY-like response regulator domains, PAS domains, GAF domains, PRD modules, and V4R domains, often represented within an N-terminal region (Fig. (Fig.1).1). These sequence features are described in more detail later in this article. Intriguingly, recent complete genome sequences have revealed some unusual EBPs containing regions of homology to other signal transduction domains and enzymes. With the large number of complete bacterial genomes now available, and with the importance of accurate annotation of future sequence data, we feel that it is timely to survey the variety of domain architectures found in these important proteins.
TL;DR: The findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes, and that Rot and agr have opposing effects on select target genes.
Abstract: Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.
TL;DR: The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.
Abstract: In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl(2) method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.
TL;DR: In the 20 years following the publication of Biochemistry of Methylotrophs, a few additional methylotrophy biochemical pathways have been discovered, such as the pathway for C1 transfer linked to methanopterin and methanofuran, which solved the long-standing mystery of formaldehyde oxidation in many methylotrophic organisms.
Abstract: Methylotrophy is defined as the ability to “grow at the expense of reduced carbon compounds containing one or more carbon atoms but containing no carbon-carbon bonds” (3). It is an intriguing example of microbial metabolic agility, with the use of a class of chemicals disregarded by the majority of organisms. Even though the ability to grow methylotrophically was first discovered in the early 1900s (cited in reference 3), it was not until the 1960s to 1970s that an understanding of the biochemical nature of this capability started to emerge. Fascination with methylotrophy in those years was fueled by the commercial interest in single-cell protein production, and as a result, the specific details of the biochemistry of methylotrophy began to be revealed. Enzymes for the primary oxidation of C1 substrates such as methanol dehydrogenase and methylamine dehydrogenase were characterized, and distinct modes of C1 assimilation, such as the ribulose monophosphate cycle and the serine cycle were discovered. The biochemical processes involved in methylotrophy that were known by 1982 are described in detail in the now classic book Biochemistry of Methylotrophs by Christopher Anthony (3). In the 20 years following the publication of Biochemistry of Methylotrophs, a few additional methylotrophy biochemical pathways have been discovered, such as the pathway for C1 transfer linked to methanopterin and methanofuran, which solved the long-standing mystery of formaldehyde oxidation in many methylotrophs (15, 53), and novel pathways for primary C1 oxidation, such as the pathways for degradation of chlorinated methanes and methanesulfonic acid (21, 50).
The knowledge concerning the biochemistry and physiology of methylotrophic organisms accumulated over the past three decades suggests a new framework for understanding methylotrophy as a novel metabolic mode. In this framework, methylotrophy is envisioned as a set of specific metabolic functional modules, with different combinations of such modules being present in different methylotrophs (Fig. (Fig.11 for the methylotrophic metabolic modules in Methylobacterium extorquens AM1). However, until recently, a number of important details of these modules were missing, and so the picture remained incomplete. The availability of two unfinished genome sequences for the important model organisms M. extorquens AM1 (http://www.integratedgenomics.com/genomereleases.html#list6)and Methylococcus capsulatus Bath (http://tigrblast.tigr.org/ufmg/) is transforming our understanding of methylotrophy. Annotation of these two genomes combined with functional analysis will delineate the set of genes and functions that is both sufficient and necessary to define a methylotroph. Expanding genomic analyses to include other groups of methylotrophs will in turn provide clues to the origins of methylotrophy and the evolution of various methylotrophic pathways. In this publication, we summarize the existing knowledge of the genes involved in methylotrophic pathways in M. extorquens AM1, analyze its yet unfinished genome with respect to location and clustering of methylotrophy genes, and present a comprehensive list of methylotrophy genes and enzymes known at this time in M. extorquens AM1 (Table (Table11).
FIG. 1.
Methylotrophy metabolic modules in M. extorquens AM1. Known genes are in italic. For simplicity, redox reactions in the assimilatory pathways are not indicated. For details, refer to the references given in Table Table11.
TABLE 1.
Methylotrophy genes in M. extorquens AM1
TL;DR: While many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway.
Abstract: The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.
TL;DR: The first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo is demonstrated and demonstrates the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.
Abstract: Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips In intrageneric coaggregation, streptococci such as Streptococcus gordonii DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as Streptococcus oralis 34 To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against S gordonii DL1 (anti-DL1) These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci Cells reactive with antibody against type 2 fimbriae of Actinomyces naeslundii T14V (anti-type-2) were much less frequent than either subset of streptococci However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque