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RcsB is the response regulator of the complex Rcs two-component system, which senses perturbations in the outer membrane and peptidoglycan layer. BglJ is a transcriptional regulator whose constitutive expression causes activation of the H-NS. and StpA-repressed bgl (aryl-[beta],D-glucoside) operon in Escherichia coli. RcsB and BglJ both belong to the LuxR-type family of transcriptional regulators with a characteristic C-terminal DNA-binding domain. Here, we show that BglJ and RcsB interact and form heterodimers that presumably bind upstream of the bgl promoter, as suggested by mutation of a sequence motif related to the consensus sequence for RcsA-RcsB heterodimers. Heterodimerization of BglJ-RcsB and relief of H-NS-mediated repression of bgl by BglJ-RcsB are apparently independent of RcsB phosphorylation. In addition, we show that LeuO, a pleiotropic LysR-type transcriptional regulator, likewise binds to the bgl upstream regulatory region and relieves repression of bgl independently of BglJ-RcsB. Thus, LeuO can affect bgl directly, as shown here, and indirectly by activating the H-NS-repressed yjjQ-bglJ operon, as shown previously. Taken together, heterodimer formation of RcsB and BglJ expands the role of the Rcs two-component system and the network of regulators affecting the bgl promoter. doi: 10.1128/JB.00807-10

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ~15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ~20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating. doi: 10.1093/icb/icq040

Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells. doi: 10.1128/JB.01266-09

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120. doi: 10.1128/JB.01668-09

The Bacillus subtilis asnH operon, comprising yxbB, yxbA, yxnB, asnH and yxaM, is induced dramatically in the transition between exponential growth and stationary phase in rich sporulation medium. The asnH operon is transcribed to produce an unstable long transcript covering the entire operon as well as a short one corresponding to the first three genes. Northern blot analysis revealed that the discrete band corresponding to the short transcript was detectable even 1 h after the addition of excess rifampicin, suggesting its unusual stability. The transcription start site of the operon was determined; its corresponding promoter was most likely sigma-A dependent and under tight control of AbrB and CodY. Within the 5'-proximal region of the transcript preceding yxbB, there is a mysterious long sequence triplication (LST) segment, consisting of a tandem repeat of two highly conserved 118 bp units and a less conserved 129 bp unit. This LST segment was not involved in regulation by AbrB and CodY. Transcriptional fusion of the 5'-region containing the LST segment to lacZ resulted in a significant increase in [beta]-galactosidase synthesis in cells; the LST segment was thought to prevent degradation of the 5'-region--lacZ fusion transcript. These results suggest that the 5'-region containing the LST segment could function as an mRNA stabilizer that prolongs the lifetime of the transcript to which it is fused. DOI 10.1099/mic.0.036582-0

It is generally known that bacterial genes working in the same biological pathways tend to group into operons, possibly to facilitate cotranscription and to provide stoichiometry. However, very little is understood about what may determine the global arrangement of bacterial genes in a genome beyond the operon level. Here we present evidence that the global arrangement of operons in a bacterial genome is largely influenced by the tendency that a bacterium keeps its operons encoding the same biological pathway in nearby genomic locations, and by the tendency to keep operons involved in multiple pathways in locations close to the other members of their participating pathways. We also observed that the activation frequencies of pathways also influence the genomic locations of their encoding operons, tending to have operons of the more frequently activated pathways more tightly clustered together. We have quantitatively assessed the influences on the global genomic arrangement of operons by different factors. We found that the current arrangements of operons in most of the bacterial genomes we studied tend to minimize the overall distance between consecutive operons of a same pathway across all pathways encoded in the genome. bacterial genome | bioinformatics | genome organization | nucleoid | neighboring genes doi/10.1073/pnas.0911237107

Excision of transposable genetic elements from host DNA occurs at low frequencies and is usually imprecise. A common insertion sequence element in Escherichia coli, IS5, has been shown to provide various benefits to its host by inserting into specific sites. Precise excision of this element had not previously been demonstrated. Using a unique system, the fucose (fuc) regulon, in which IS5 insertion and excision result in two distinct selectable phenotypes, we have demonstrated that IS5 can precisely excise from its insertion site, restoring the wild-type phenotype. In addition to precise excision, several 'suppressor' insertion, deletion, and point mutations restore the wild-type [Fuc.sup.+] phenotype to various degrees without IS5 excision. The possible bases for these observations are discussed. doi: 10.1128/JB.01085-09

Bacterial two-hybrid studies of randomly cloned Escherichia coli DNA identified a physical interaction between GyrA, subunit A of gyrase, and MarR, a repressor of the marRAB operon. GyrA-His immobilized on Ni-nitrilotriacetic acid (NiNTA) resin bound MarR, while MarR alone did not bind. GyrA interfered with MarR binding to marO, as detected by electrophoretic mobility assays. In a strain bearing the marRAB operon and a marO-lacZ reporter, overexpression of GyrA increased LacZ activity, indicating decreased repression of marO-lacZ by MarR. These results were confirmed by an increased survival of cells treated with quinolones and other antibiotics when GyrA was overexpressed. This work, like a previous study examining TktA (12), shows that unrelated proteins can regulate MarR activity. The findings reveal an unexpected regulatory function of GyrA in antibiotic resistance. doi: 10.1128/JB.01259-09

The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTX[PHI]. Usually, specific CTX[PHI] infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called [CTX.sup.class][PHI] and [CTX.sup.ET][PHI], respectively. However, an unusual hybrid El Tor strain carrying [CTX.sup.class][PHI] caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique [CTX.sup.class][PHI] having only four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3' present in the ctxAB promoter ([P.sub.ctxAB]) region, like in [CTX.sup.ET][PHI]. Usually, the [P.sub.ctxAB] of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the [P.sub.ctxAB] regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes. DOI 10.1099/mic.0.032458-0

A unique D-to-L racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon has been recently reported as a prerequisite for D-arginine utilization as the sole source of carbon and nitrogen through L-arginine catabolic pathways in P. aeruginosa. In this study, enzymic properties of the catabolic FAD-dependent D-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other D-amino acids. These results establish DauA as a D-amino acid dehydrogenase of broad substrate specificity, with D-Arg and D-Lys as the two most effective substrates, based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous D-Arg and D-Lys, and mutations in the dauBAR operon affect utilization of these two amino acids alone. The function of DauR as a repressor in the control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from D-Arg deamination by DauA as a signal molecule in dauBAR induction was first revealed by mutation analysis and further supported by its in vitro effect on alleviation of DauR-DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the L-arginine-responsive regulator ArgR, as supported by the loss of inductive effect by LArg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study establishes that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by L-Arg as a result of D-Arg racemization by the encoded DauA and DauB. DOI 10.1099/mic.0.033282-0

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites. doi: 10.1128/JB.01000-09

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in grampositive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The [[DELTA]dlt.sub.Bc] mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodopterafrugiperda. [[DELTA]dlt.sub.Bc] was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. meUonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in [[DELTA]dlt.sub.Bc]). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans. doi: 10.1128/JB.00892-09

Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP. These residues are conserved in TnaC of P. vulgaris and of other bacterial species. TnaC of P. vulgaris has one additional feature, distinguishing it from TnaC of E. coli; it contains two C-terminal lysine residues following the conserved proline residue. In the present study, we investigated L-tryptophan induction of the P. vulgaris tna operon, transferred on a plasmid into E. coli. Induction was shown to be L-tryptophan dependent; however, the range of induction was less than that observed for the E. coli tna operon. We compared the genetic organization of both operons and predicted similar folding patterns for their respective leader mRNA segments. However, additional analyses revealed that L-tryptophan action in the P. vulgaris tna operon involves inhibition of TnaC elongation, following addition of proline, rather than inhibition of leader peptide termination. Our findings also establish that the conserved residues in TnaC of P. vulgaris are essential for L-tryptophan induction, and for inhibition of peptide elongation. TnaC synthesis is thus an excellent model system for studies of regulation of both peptide termination and peptide elongation, and for studies of ribosome recognition of the features of a nascent peptide. doi: 10.1128/JB.01002-09

Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments. doi: 10.1128/JB.00450-09

The asc operon of Escherichia coli is one of the cryptic genetic systems for [beta]-D-galactoside utilization as a carbon source. The ascFB genes for [beta]-D-galactoside transport and catabolism are repressed by the AscG regulator. After genomic SELEX screening, AscG was found to recognize and bind the consensus palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites upstream of the ascFB promoter and at three sites upstream of the prpBC operon for propionate catabolism. In an ascG-disrupted mutant, transcription of ascFB was enhanced, in agreement with the repressor model of AscG. This repression was indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box, which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase to the promoter. Under conditions of steady-state E. coli growth in a rich medium, the intracellular level of AscG stayed constant at a level supposedly leading to tight repression of the ascFB operon. The level of prpR, encoding the activator of prpBCDE, was also increased in the absence of AscG, indicating the involvement of AscG in repression of prpR. Taken together, these data suggest a metabolic link through interplay between the asc and prp operons. doi: 10.1128/JB.00663-09

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-[N.sub.11]-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxR'A'::[OMEGA] toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.

On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GAIT, GaIK and GaIE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80[degrees]C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

Previous transcript mapping of the bacteriophage Aeh1 nrd operon revealed a predicted RNA hairpin upstream of the homing endonuclease mobE gene. We enzymatically mapped the hairpin, showing that the mobE ribosome binding site is sequestered. Cloning of the hairpin upstream of lacZ resulted in reduced [beta]-galactosidase activity, consistent with translational regulation.

The Bacillus subtilis genome has been sequenced, and disruptants with disruptions in genes that were not characterized previously were systematically generated. We screened these gene disruptants for decreased transformation frequency and identified two genes, yrzD and yutB, whose disruption resulted in severely reduced transformation frequency and modestly reduced transformation frequency, respectively. In the regulation of competence development, various signals affect the expression of comK, which encodes a master regulator of genetic competence that drives late competence gene transcription. Epistatic analyses of both the yrzD and yutB genes revealed no significant differences in the expression of comK. Further analysis of the expression of late competence genes in the yrzD disruptant revealed that yrzD is specifically required for regulation of the comE operon, which is one of the late competence operons, and thus was renamed comN. An analysis of various comE-lacZ fusions revealed that the target cis element for comN action is in the large (approximately 1-kb) 5' untranslated region of comE, while the activity of the comE promoter was not affected by disruption of comN. These results suggested that there is post-transcription initiation control of comE by comN. A sequential deletion analysis of this region revealed the 35-bp region required for comN action. The yutB gene encodes a putative lipoic acid synthetase and yet is specifically required for transcription of comE, based on the results of lacZ fusion analyses. Therefore, yutB and comN regulate comE at the transcription and post-transcription initiation levels, respectively. These results demonstrate that a comE-specific regulatory mechanism is involved in development of genetic competence.

RNA polymerase II (Pol II) transcription termination involves two linked processes: mRNA 3'-end formation and release of Pol II from DNA. Signals for 3' processing are recognized by a protein complex that includes cleavage polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF). Here we identify suppressors encoding proteins that play roles in processes at the 3' ends of genes by exploiting a mutation in which the 3' end of another gene is transposed into the first gene of the Caenorhabditis elegans lin-15 operon. As expected, genes encoding CPSF and CstF were identified in the screen. We also report three suppressors encoding proteins containing a domain that interacts with the C-terminal domain of Pol II (CID). We show that two of the CID proteins are needed for efficient 3' cleavage and thus may connect transcription termination with RNA cleavage. Furthermore, our results implicate a serine/arginine-rich (SR) protein, SRp20, in events following 3'-end deavage, leading to termination of transcription. Caenorhabditis elegans operon | RNA polymerase II C-terminal domain | SRp20 | CTD

Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to [10.sup.5]-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).

The recent sequence analysis of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain BTAi1 revealed the unexpected presence of a pucBA operon encoding the apoproteins of peripheral light-harvesting (LH) complexes. This pucBA operon is found close to a bacteriophytochrome gene ([BphP3.sub.B BTAi1]) and a two-component transcriptional regulator gene ([TF.sub.BTAi1] gene). In this study, we show that [BphP3.sub.B BTAi1] acts as a bona fide bacteriophytochrome and controls, according to light conditions, the expression of the pucBA operon found in its vicinity. This light regulatory pathway is very similar to the one previously described for chromo-[BphP4.sub.RP] in Rhodopseudomonas palustris and conducts the synthesis of a peripheral LH complex. This LH complex presents a single absorption band at low temperature, centered at 803 nm. Fluorescence emission analysis of intact cells indicates that this peripheral LH complex does not act as an efficient light antenna. One putative function of this LH complex could be to evacuate excess light energy in order to protect Bradyrhizobium strain BTAi1, an aerobic anoxygenic photosynthetic bacterium, against photooxidative damage during photosynthesis.

Trimethylamine N-oxide (TMAO) reductases are widespread in bacteria and often function in anaerobic respiration. The regulation and expression of TMAO reductase operons have been well studied in model genera such as Escherichia, Shewanella, and Rhodobacter, although TMAO reductases are present in many other bacteria, including the marine Vibrio species. The genome sequence of Vibrio fischeri revealed three putative TMAO reductase operons, and a previous report identified TMAO reductase activity in symbiotic V. fischeri isolates associated with the light organs of adult Hawaiian bobtail squid, Euprymna scolopes. We examined the roles and regulation of these three operons using mutational analyses and promoter-reporter fusions. We found that the torECA promoter, and to a lesser extent the torYZ and dmsABC promoters, were active during symbiotic colonization of juvenile E. scolopes; however, a V. fischeri strain lacking TMAO reductase activity displays no discernible colonization defect over the first 48 h. Our studies also revealed that torECA has the most active promoter of the putative TMAO reductase operons, and TorECA is the major contributor to TMAO-dependent growth in V. fischeri under the conditions tested. Interestingly, the transcriptional regulation of TMAO reductase operons in V. fischeri appears to differ from that in previously studied organisms, such as Escherichia coli, which may reflect differences in gene arrangement and bacterial habitat. This study lays the foundation for using V. fischeri as a model system for studying TMAO reductases in the Vibrionaceae.

The effect of bulk-phase pH on the apparent affinity ([K.sup.app.sub.d]) of purified wild-type lactose permease (LacY) for sugars was studied. [K.sup.app.sub.d] values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. [K.sup.app.sub.d] for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high [pK.sub.a] of [approximately equal to] 10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/[H.sup.+] symport in either direction across the membrane. lactose permease | membrane transporters | pH titrations | proton translocation | substrate affinity

Erwinia carotovora subsp, carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant ceil wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhD[C.sup.-] mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin ([Harpin.sub.Ecc]) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhD[C.sup.+] plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp, carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhD[C.sup.-] mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an Rsm[A.sup.-] FlhD[C.sup.-] double mutant and multiple copies of rsm[B.sup.+] DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp, carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp, carotovora.

Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a [sigma]54-dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNA-binding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although [CySB.sub.Ds1] appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA-binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo by using the disruptants of the sulfate assimilatory genes cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested that the expression of sfnFG is repressed by sulfate itself while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.

The fim operon of Salmonella enterica serovar Typhimurium encodes type 1 fimbriae. The expression of fim is controlled in response to environmental signals through a complex regulatory cascade involving the proteins FimW, FimY, and FimZ and a genetic locus, fimU, that encodes a rare arginine tRNA. We discovered that a knockout mutation in lrp, the gene that codes for the leucine-responsive regulatory protein (Lrp), inhibited fim transcription. The loss of tim gene expression was accompanied by a corresponding loss of the mannosesensitive hemagglutination that is a characteristic of type 1 fimbriae. Normal type 1 fimbrial expression was restored following the introduction into the knockout mutant of a plasmid carrying a functional copy of the lrp gene. Electrophoretic mobility shift analysis revealed no interactions between purified Lrp protein and the regulatory region of the fimA, fimU, or fimW gene. Instead, Lrp produced protein-DNA complexes with the regulatory region of the fimZ gene, and the nature of these complexes was leucine sensitive. DNase I foot-printing showed that Lrp binds within a region between -65 and -170 with respect to the fimZ transcription start site, consistent with the binding and wrapping of the DNA in this upstream region. Ectopic expression of the fimZ gene from an inducible promoter caused Lrp-independent type I fimbriation in serovar Typhimurium. These data show that Lrp makes a positive contribution to fim gene expression through direct interaction with the fimZ promoter region, possibly by antagonizing the binding of the H-NS global repressor protein.

We constructed monocopy lac operon control regions in which the operators O1-lac and O3-lac were replaced by NarL and NarP binding sites from the nirB or napF operon control regions. The results support the hypothesis that DNA-bound dimers of phospho-NarL can participate in higher-order cooperative interactions.

Many species of bacteria can adhere to surfaces and grow as sessile communities. The continued accumulation of bacteria can eventually lead to the extremely high-cell-density environment characteristic of many biofilms or cell colonies. This is the normal habitat of the cariogenic species Streptococcus mutans, which normally resides in the high-cell-density, multispecies community commonly referred to as dental plaque. Previous work has demonstrated that the transcription of two separate bacteriocins can be activated by the high-cell-density conditions created through the centrifugation and incubation of cell pellets. In this study, we identified an uncharacterized two-gene operon that was induced >10-fold by conditions of high cell density. The genes of the operon encode a putative transcription regulator and a membrane protein, which were renamed as hdrR and hdrM, respectively. A transcription fusion to the hdrRM operon confirmed its induction by high cell density. Mutation of hdrM abolished bacteriocin production, greatly increased natural competence, reduced the growth rate, and severely affected biofilm formation. Interestingly, no obvious phenotypes were observed from a non-polar mutation of hdrR or mutations affecting the entire operon. These data suggest that the hdrRM operon may constitute a novel regulatory system responsible for mediating a cellular response to a high-cell-density environment.

Vibrio parahaemolyticus is a marine bacterium bearing 11 copies of ribosomal operons. In some strains, such as RIMD2210633, the genome includes identical copies of 16S rRNA genes (rrs). However, it is known that other strains of the species, such as strains ATCC 17802 and RIMD2210856, show conspicuous intragenomic rrs heterogeneity. The extent and diversity of the rrs heterogeneity in V. parahaemolyticus were studied in further detail by characterization of the rrs copies in environmental isolates belonging to 21 different genotype groups. Thirteen of these groups showed intragenomic heterogeneity, containing altogether 16 sequences differing within a 25 bp segment of their rrs. These sequences grouped into four clusters differing in at least four nucleotide sites. Some isolates contained rrs alleles from up to three different clusters. Each segment sequence conserved the stem-loop characteristic of the 16S rRNA structure of this 25 bp sequence. The double-stranded stem sequence was quite variable, but almost every variation had a compensatory change to maintain seven to eight paired bases. Conversely, the single-strand loop sequence was conserved. The results may be explained as a consequence of recombination among rrs evolving in different bacteria. The results suggest that intergenomic rrs recombination is very high in V. parahaemolyticus and that it occurs solely among Vibrio species. This high rrs homologous intergenomic recombination could be an effective mechanism to maintain intragenomic rrs cohesion, mediating the dispersal of the most abundant rrs version among the 11 intragenomic loci.

We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-[N.sub.6]-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adhl, lctE, srrAB, and lukM. In one case, for lctE, encoding L-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A, actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.

Retraction of type IV pili is mediated by PilT. We show that loss of pilT function leads to upregulation of mtrF (multiple transferable resistance) and two operons encoding putative ABC transporters in Neisseria gonorrhoeae MS11. This effect occurs indirectly through the transcriptional regulator FarR, which until now has been shown to regulate only farAB. L-Glutamine can reverse pilT downregulation of the ABC transporter operons and mtrF.

The genome sequences of several pseudomonads have revealed a gene cluster containing genes for a two-component heavy metal histidine sensor kinase and response regulator upstream of cinA and cinQ, which we show herein to encode a copper-containing azurin-like protein and a pre-[Q.sub.0] reductase, respectively. In the presence of copper, Pseudomonas putida KT2440 produces the CinA and CinQ proteins from a bicistronic mRNA. UV-visible spectra of CinA show features at 439, 581, and 719 nm, which is typical of the plastocyanin family of proteins. The redox potential of the protein was shown to be 456 [+ or -] 4 mV by voltametric titrations. Surprisingly, CinQ is a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the conversion of pre-[Q.sub.0] to pre-[Q.sub.1] in the nucleoside queuosine biosynthetic pathway. Gene disruptions of cinA and cinQ did not lead to a significant increase in the copper sensitivity of P. putida KT2440 under the conditions tested. Possible roles of CinA and CinQ to help pseudomonads adapt and survive under prolonged copper stress are discussed.

Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the ImrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the ImrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter [beta]-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.

Oligopeptides internalized by oligopeptide permease (Opp) transporters play key roles in bacterial nutrition, signaling, and virulence. To date, two opp operons, opp-1 and opp-2, have been identified in Staphylococcus aureus. Systematic in silico analysis of 11 different S. aureus genomes revealed the existence of two new opp operons, opp-3 and opp-4, plus an opp-5A gene encoding a putative peptide-binding protein. With the exception of opp-4, the opp operons were present in all S. aureus strains. Within a single strain, the different opp operons displayed little sequence similarity and distinct genetic organization. Transcriptional studies showed that opp-1, opp-2, opp-3, and opp-4 operons were polycistronic and that opp-5A is monocistronic. We designed a minimal chemically defined medium for S. aureus RN6390 and showed that all opp genes were expressed but at different levels. Where tested, OppA protein production paralleled transcriptional profiles, opp-3, which encodes proteins most similar to known peptide transport proteins, displayed the highest expression level and was the only transporter to be regulated by specific amino acids, tyrosine and phenylalanine. Defined deletion mutants in one or several peptide permeases were constructed and tested for their capacity to grow in peptide-containing medium. Among the four putative Opp systems, Opp-3 was the only system able to provide oligopeptides for growth, ranging in length from 3 to 8 amino acids. Dipeptides were imported exclusively by DtpT, a proton-driven di- and tripeptide permease. These data provide a first complete inventory of the peptide transport systems opp and dtpT of S. aureus. Among them, the newly identified Opp-3 appears to be the main Opp system supplying the cell with peptides as nutritional sources.

The virR operon, located on the virulence plasmid of the intracellular pathogen Rhodococcus equi, contains five genes, two of which (virR and orf8) encode transcriptional regulators. The first gene of the operon (virR), encoding a LysR-type transcriptional regulator, is transcribed at a constitutive low level, whereas the four downstream genes are induced by low pH and high growth temperature. Differential regulation of the virR operon genes could not be explained by differential mRNA stability, as there were no major differences in mRNA half-lives of the transcripts representing each of the five genes within the virR operon. Transcription of virR is driven by the [P.sub.virR] promoter, with a transcription start site 53 bp upstream of the virR initiation codon. The four genes downstream of virR are transcribed from [P.sub.virR] and from a second promoter, [P.sub.orf5, located 585 bp downstream of the virR initiation codon. VirR binds to a site overlapping the initiation codon of virR, resulting in negative autoregulation of the virR gene, explaining its low constitutive transcription level. The [P.sub.orf5] promoter is induced by high temperature and low pH, thus explaining the observed differential gene expression of the virR operon. VirR has a positive effect on [P.sub.orf5] activity, whereas the response regulator encoded by orf8 is not involved in regulating transcription of the virR operon. The [P.sub.virR] promoter is strikingly similar to those recognized by the principal sigma factors of Streptomyces and Mycobacterium, whereas the [P.sub.orf5] promoter does not share sequence similarity with [P.sub.virR]. This suggests that Porfs is recognized by an alternative sigma factor.

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing [DELTA]ybxF, [DELTA]ymxC, or [DELTA]ybxF AymxC double deletion strains in several functional assays.

Lactobacillus acidophilus NCFM is an industrially important strain used extensively as a probiotic culture. Tolerance of the presence of bile is an attribute important to microbial survival in the intestinal tract. A whole-genome microarray was employed to examine the effects of bile on the global transcriptional profile of this strain, with the intention of elucidating genes contributing to bile tolerance. Genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. A 7-kb eight-gene operon encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins was significantly upregulated in the presence of bile. Deletion mutations were constructed in six genes of the operon. Transcriptional analysis of the 2CRS mutants showed that mutation of the histidine protein kinase (HPK) had no effect on the induction of the operon, whereas the mutated response regulator (RR) showed enhanced induction when the cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance and that the operon it resides in is negatively controlled by the RR. Mutations in the transporter, the HPK, the RR, and a hypothetical protein each resulted in loss of tolerance of bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. This functional analysis showed that the operon encoded proteins involved in both bile tolerance and bile sensitivity.

The Lyme disease spirochaete, Borrelia burgdorferi, produces the LuxS enzyme both in vivo and in vitro; this enzyme catalyses the synthesis of homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD) from a by-product of methylation reactions. Unlike most bacteria, B. burgdorferi is unable to utilize homocysteine. However, DPD levels alter expression levels of a subset of B. burgdorferi proteins. The present studies demonstrate that a single B. burgdorferi operon encodes both of the enzymes responsible for synthesis of DPD, as well as the enzyme for production of the Lyme spirochaete's only activated-methyl donor and a probable phosphohydrolase. Evidence was found for only a single transcriptional promoter, located 5' of the first gene, which uses the housekeeping [[sigma].sup.70] subunit for RNA polymerase holoenzyme function. All four genes are co-expressed, and mRNA levels are growth-rate dependent, being produced during the exponential phase. Thus, high metabolic activity is accompanied by increased cellular levels of the only known borrelial methyl donor, enhanced detoxification of methylation by-products, and increased production of DPD. Therefore, production of DPD is directly correlated with cellular metabolism levels, and may thereby function as an extracellular and/or intracellular signal of bacterial health.

In lactic acid bacteria (LAB), amino acids and their derivatives may be converted into amine-containing compounds designated biogenic amines, in pathways providing metabolic energy and/ or acid resistance to the bacteria. In a previous study, a pathway converting tyrosine to tyramine was detected in Lactobacillus brevis and a fragment of a gene possibly involved in the production of another biogenic amine, putrescine, from agmatine, was detected in the same locus. The present study was carried out to determine if Lb. brevis actually harbours two biogenic amine-producing pathways in the same locus and to investigate the occurrence of the two gene clusters in other bacteria. Sequencing of the DNA locus in Lb. brevis revealed a cluster of six genes that are related to previously reported genes of agmatine deiminase pathways but with marked differences such as two genes encoding putative agmatine deiminases rather than one. Heterologous expression of encoded enzymes confirmed the presence of at least one active agmatine deiminase and one amino acid transporter that efficiently exchanged agmatine and putrescine. It was concluded that the Lb. brevis gene cluster encodes a functional and highly specific agmatine deiminase pathway. Screening of a collection of 197 LAB disclosed the same genes in 36 strains from six different species, and almost all the positive bacteria also contained the tyrosine catabolic pathway genes in the same locus. These results support the hypothesis that the agmatine deiminase and tyrosine catabolic pathways belong to a genomic region that provides acid resistance and that is exchanged horizontally as a whole between LAB.

The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position -40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR reguion in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.

The nucleotide sequence of the linear catabolic plasmid pAL1 from the 2-methylquinoline (quinaldine)-degrading strain Arthrobacter nitroguajacolicus Ru61a comprises 112,992 bp. A total of 103 open reading frames (ORFs) were identified on pAL1, 49 of which had no annotatable function. The ORFs were assigned to the following functional groups: (i) catabolism of quinaldine and anthranilate, (ii) conjugation, and (iii) plasmid maintenance and DNA replication and repair. The genes for conversion of quinaldine to anthranilate are organized in two operons that include ORFs presumed to code for proteins involved in assembly of the quinaldine-4-oxidase holoenzyme, namely, a MobA-like putative molybdopterin cytosine dinucleotide synthase and an XdhC-like protein that could be required for insertion of the molybdenum cofactor. Genes possibly coding for enzymes involved in anthranilate degradation via 2-aminobenzoyl coenzyme A form another operon. These operons were expressed when cells were grown on quinaldine or on aromatic compounds downstream in the catabolic pathway. Single-stranded 3' overhangs of putative replication intermediates of pAL1 were predicted to form elaborate secondary structures due to palindromic and superpalindromic terminal sequences; however, the two telomeres appear to form different structures. Sequence analysis of ORFs 101 to 103 suggested that pAL1 codes for one or two putative terminal proteins, presumed to be covalently bound to the 5' termini, and a multidomain telomere-associated protein (Tap) comprising 1,707 amino acids. Even if the putative proteins encoded by ORFs 101 to 103 share motifs with the Tap and terminal proteins involved in telomere patching of Streptomyces linear replicons, their overall sequences and domain structures differ significantly. doi:10.1128/JB.00089-07

Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its -35 and -10 regions resemble those recognized by M. xanthus [[sigma].sup.A] RNA polymerase, the homolog of Escherichia coli [[sigma].sup.70], but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus. doi:10.1128/JB.00187-07

Bacillus subtilis has multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containing ssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for the secG-yvaK-rnr-smpB-ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One [[sigma].sup.A]-like promoter preceded secG ([P.sub.1]), and internal [[sigma].sup.A]-like promoters were found in both the rnr.smpB ([P.sub.2]) and smpB-ssrA intervals ([P.sub.3] and [P.sub.HS]). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity ([P.sub.B]) was dependent on the general stress factor [[sigma].sup.B]. Null mutations in the four genes downstream from [P.sub.B] were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was required for growth at high temperature (52[degrees]C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably, smpB and ssrA were also required for growth at low temperature (16[degrees]C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range. doi:10.1128/JB.00062-.07

Desulfovibrio desulfuricans G20 grows and reduces 20 mM arsenate to arsenite in lactate-sulfate media. Sequence analysis and experimental data show that D. desulfuricans G20 has one copy of arsC and a complete arsRBCC operon in different locations within the genome. Two mutants of strain G20 with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. The arsRBCC operons were intact in both mutant strains, but each mutant had one point mutation in the single arsC gene. Mutants transformed with either the arsC1 gene or the arsRBCC operon displayed wild-type arsenate resistance, indicating that the two arsC genes were equivalently functional in the sulfate reducer. The arsC1 gene and arsRBCC operon were also cloned into Escherichia coli DH5[alpha] independently, with either DNA fragment conferring increased arsenate resistance. The recombinant arsRBCC operon allowed growth at up to 50 mM arsenate in LB broth. Quantitative PCR analysis of mRNA products showed that the single arsC1 was constitutively expressed, whereas the operon was under the control of the arsR repressor protein. We suggest a model for arsenate detoxification in which the product of the single arsC1 is first used to reduce arsenate. The arsenite formed is then available to induce the arsRBCC operon for more rapid arsenate detoxification. doi:10.1128/JB.01913-06