A Neanderthal lower molar from Stajnia Cave, Poland.

The primary aim of this study was to conduct a taxonomic assessment of the second of three isolated human teeth found in the Stajnia Cave (north of the Carpathians, Poland) in 2008. The specimen was located near a human tooth (S5000), which was identified by Urbanowski et al. (2010) as a Neanderthal permanent upper molar. Both of these teeth were excavated from the D2 layer, which belongs to the D stratigraphic complex comprising the archaeological assemblage associated with the Micoquian tradition. An Ursus spelaeus bone and Mammuthus primigenius tooth that were also excavated from the D2 layer were dated to >49,000 years BP (by AMS (14)C) and 52.9 ka BP (by U-Th), respectively. The sediment overlying stratigraphic complex D was dated to 45.9 ka BP by the OSL method. The S4300 tooth is a lower first or second permanent molar belonging to an individual other than that who once possessed the S5000 tooth. The S4300 tooth exhibits a combination of traits typical of Neanderthal lower molars, including a mid-trigonid crest, large anterior fovea, taurodontism and subvertical grooves on the interproximal face, indicating that this tooth belonged to a Neanderthal individual. The S4300 tooth from Stajnia Cave is one of the oldest human remains found in Poland.

Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners.

Vibrio fischeri belongs to the Vibrionaceae, a large family of marine gamma-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.

Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR.

Along with their cognate acyl-homoserine lactone signals, the quorum sensing regulators LasR and RhlR control the expression of hundreds of genes in the opportunistic human pathogen Pseudomonas aeruginosa. This extensive, overlapping regulatory network affords the opportunity to systematically investigate the sequence requirements and specificity determinants of large families of target promoters. Many of the P. aeruginosa quorum-controlled genes possess conserved palindromic promoter elements predicted to be binding sites for either one or both transcriptional regulators, but biochemical proof has not been reported. We have purified native LasR and characterized binding to various quorum-controlled promoters in vitro. Purified LasR was a dimer in solution that irreversibly bound two molecules of 3-oxo-C12-homoserine lactone. LasR bound several las-responsive promoters specifically and with high affinity, interacting cooperatively with some promoters and noncooperatively with others. LasR recognized some, but not all, of the predicted binding sites, and also bound to several unexpected sites. In contrast to predictions from genetic data, we found that the recognition sequences of las-specific promoters showed little overall sequence conservation and did not require dyad symmetry. We found distinct differences in sequence composition between las-specific noncooperative, las-specific cooperative, and rhl-responsive promoters. These results provide the basis for defining promoter specificity elements in P. aeruginosa quorum sensing. Insights into the molecular mechanism of LasR function have implications for the development of quorum-sensing targeted antivirulence compounds.

Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein.

The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors. Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon. We have purified LuxR from recombinant Escherichia coli. Purified LuxR binds specifically and with high affinity to DNA containing a lux box. This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex. When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E. coli RNA polymerase to initiate transcription from the luxI promoter. Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent. Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different. The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density.

Role of the RNA polymerase alpha subunits in MetR-dependent activation of metE and metH: important residues in the C-terminal domain and orientation requirements within RNA polymerase.

Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP alpha subunit (alphaCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify alphaCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the alphaCTD. The first is a complex surface consisting of residues important for alpha-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of alpha required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant alpha subunits to either the beta-associated or the beta'-associated position within RNAP (comprising alpha(2), beta, beta', and sigma subunits). In vitro transcriptions using oriented alpha RNAP indicate that a single alphaCTD on either the beta- or the beta'-associated alpha subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the alphaCTD on the beta-associated alpha subunit at metH. We propose that the different alphaCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the alphaCTD.

The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli.

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic-binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA-phoA and dppA-lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli.

A mutation in the rpoA gene (which encodes the alpha subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a lambda metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the -35 region of the metE promoter that interacts with the alpha subunit. Our data indicate that the glutamic acid at position 261 in the alpha subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.

DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli.

The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA. Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions. DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from -28 to +20. Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions -271 to -242, a 28-bp region extending from -242 to -214, and a 35-bp region covering positions -69 to -34 relative to the transcription initiation site. PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv.

Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium.

Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator. It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides -48 to -71 relative to the metE transcription initiation site (designated as site 1). In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides -24 to -47 relative to the metE transcription initiation site (designated as site 2). Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion. Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression. DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2. A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.

A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli.

The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli. However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase. In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the alpha subunit of RNA polymerase that decreases metE expression independently of the MetR protein. The mutation does not affect expression from the metH promoter, suggesting that the alpha subunit of RNA polymerase interacts differently at these two promoters. The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue. Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression. This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the alpha subunit in sequence-specific recognition of the metE promoter.

Regulation of the Salmonella typhimurium metF gene by the MetR protein.

The metF gene in Escherichia coli and Salmonella typhimurium is under negative transcriptional control by the MetJ repressor. Expression of an S. typhimurium metF-lacZ gene fusion is repressed up to 10-fold by methionine addition to the growth medium in E. coli hosts encoding wild-type MetJ repressor; this repression is not seen in metJ mutants. metR mutations which eliminate the MetR activator protein result in two- to threefold-more-severe repression by the MetJ repressor. In a metJ metR double mutant, however, the level of metF-lacZ expression is the same as in a metJ mutant, suggesting that MetR antagonizes MetJ-mediated methionine repression of the metF promoter. A DNA footprint analysis showed that MetR binds to a DNA fragment carrying the metF promoter and protects two separate regions from DNase I digestion: a 46-bp region from position -50 to -95 upstream of the transcription initiation site and a 24-bp region from about position +62 to +85 downstream of the transcription initiation site and within the metF structural gene. Nucleotide changes in each of the MetR-binding sites away from the consensus sequence disrupt MetR-mediated regulation of the metF-lacZ fusion.

MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region.

In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed. Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the -35 region of the metR promoter. Starting with a metE-lacZ.metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence. Each mutation, along with the wild-type metE-lacZ.metR-galK gene fusion, was cloned into phage lambda gt2. Regulation of the metE and metR genes was examined by measuring beta-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions. Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene.

The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme.

Regulation of the Salmonella typhimurium metA gene by the metR protein and homocysteine.

The DNA sequence of the Salmonella typhimurium metA control region is presented. S1 nuclease mapping was used to determine the transcription initiation site. By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene. Homocysteine, an intermediate in methionine biosynthesis, plays a negative role in the MetR-mediated activation mechanism. Gel mobility shift assays and DNase I protection experiments showed that the MetR protein binds to a DNA fragment carrying the metA control region and protects a 26-bp region beginning 9 bp upstream of the -35 promoter sequence.

The metR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation.

Transcription of the metH gene in Salmonella typhimurium and Escherichia coli is positively regulated by the metR gene product, a DNA binding protein. The interaction between the MetR activator protein and the S. typhimurium metH control region was investigated. In vitro gel mobility shift assays and DNase I protection assays established that the MetR protein binds to and protects a 24-bp sequence in the metH promoter region from DNase I attack. This region includes the proposed metR recognition sequence 5'-TGAANNNNNCTCA-3'. Single-base-pair changes were introduced into the proposed MetR recognition sequence within the promoter region of a metH-lacZ gene fusion by oligonucleotide-directed mutagenesis. Two classes of mutations were identified. In the first class, the mutations caused reduced activation of the metH-lacZ fusions that correlated with reduced MetR binding. In the second class, activation of the metH-lacZ fusion was reduced, yet there was no appreciable reduction in MetR binding, indicating that the presence of bound MetR is not sufficient for activation of metH-lacZ gene expression. These two classes of mutations in the DNA binding site are grouped spatially, suggesting that the proposed MetR recognition sequence can be divided into two functional domains, one for binding and the other for activation.

Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response.

Using an Escherichia coli lac deletion strain lysogenized with a lambda phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression.

Nucleotide sequence of the Salmonella typhimurium glyA gene.

The DNA sequence of the Salmonella typhimurium glyA gene has been determined. The polypeptide deduced from the DNA sequence contains 417 amino acids and has a calculated molecular weight of 45428 daltons. S1 nuclease mapping experiments located the transcription start point and possible transcription termination region. The nucleotide and amino acid sequences for the S. typhimurium and Escherichia coli glyA genes were compared. The nucleotide sequences show 89% identity, and the amino acid sequences show 93% identity. In S. typhimurium there is an absence of REP sequences between the translation termination site and the proposed transcription termination site that are present in the E. coli sequence. A conserved sequence is found in both organisms extending from 79 to 117 bp upstream of the consensus -35 sequences of the glyA promoters. This conserved sequence shows homology to a sequence preceding the S. typhimurium metE gene determined to bind the MetR regulatory protein.

Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium.

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.

Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli.

The metR-mediated activation of the Salmonella typhimurium metE and metH genes was shown to be modulated by homocysteine, an intermediate in the methionine biosynthetic pathway. Homocysteine stimulates expression of a metE-lacZ gene fusion four- to fivefold by increasing transcription from the metE promoter. In contrast, homocysteine plays an inhibitory role in the metR-mediated activation of the metH gene, decreasing expression of a metH-lacZ gene fusion threefold.

The control region of the metH gene of Salmonella typhimurium LT2: an atypical met promoter.

The control region of the Salmonella typhimurium metH gene was sequenced and the transcription start point was determined by S1 nuclease mapping experiments. Activation of the metH gene by the metR gene product was shown to occur at the level of transcription. The translation start site was determined by amino acid sequence analysis of the amino terminus of a chimeric Met-Lac fusion protein encoded by a metH-lacZ gene fusion. Analysis of the nucleotide sequence of the metH promoter region showed that two sequence elements, present in the promoters of all other met biosynthetic genes thus far examined, are not present in the metH promoter region, namely, the repeated MetJ repressor recognition sequence 5'-AGACGTCT-3' and a highly conserved sequence 5'-TGGA----TAAAC-3' of unknown function.