Isolation and draft genome sequence of Enterobacter asburiae strain i6 amenable to genetic manipulation.

Enterobacter asburiae is a species of Gram-negative bacteria that is found in soil, water, and sewage. E. asburiae is generally considered to be an opportunistic pathogen, but has also been reported as a plant growth-promoting bacterium (PGPB), which may have beneficial effects on plant growth and development. However, genetic analysis of E. asburiae has been limited, possibly due to its redundant enzymes that digest exogenous DNA in the cell. Here, an E. asburiae strain i6 was isolated from soil in Nara, Japan. This strain was amenable to transformation and the one-step gene inactivation method based on λ Red recombinase. The transformation efficiency of the i6 strain with the 10 kb plasmid DNA pCF430 was at least four orders of magnitude higher than that of the previously sequenced E. asburiae strain ATCC 35953, which could not be transformed with the same plasmid DNA. A draft genome sequence of the i6 strain was determined and deposited into the database, allowing several factors that may determine transformation efficiency to be perturbed and tested. Together with the amenability of the i6 strain to genetic manipulation, the information from the i6 genome will facilitate characterization and fine-tuning of the beneficial and detrimental traits of this species.

Development of conjugation-mediated versatile site-specific single-copy luciferase fusion system.

There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and KmR marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.

Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control.

The PhoQ/PhoP two-component signal transduction system is conserved in various Gram-negative bacteria and is often involved in the expression of virulence in pathogens. The small inner membrane protein SafA activates PhoQ in Escherichia coli independently from other known signals that control PhoQ activity. We have previously shown that SafA directly interacts with the sensor domain of the periplasmic region of PhoQ (PhoQ-SD) for activation, and that a D179R mutation in PhoQ-SD attenuates PhoQ activation by SafA. In this study, structural comparison of wild-type PhoQ-SD and D179R revealed a difference in the cavity (SD (sensory domain) pocket) found in the central core of this domain. This was the only structural difference between the two proteins. Site-directed mutagenesis of the residues surrounding the SD pocket has supported the SD pocket as a site involved in PhoQ activity. Furthermore, the SD pocket has also been shown to be involved in SafA-mediated PhoQ control.

Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis.

The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.

Fe3+-dependent epistasis between the CpxR-activated loci and the PmrA-activated LPS modification loci in Salmonella enterica.

Bacteria utilize varying combinations of two-component regulatory systems, many of which respond and adapt closely to stress conditions, thus expanding their niche steadily. While mechanisms of recognition and avoidance of the specific Fe3+ signal by the PmrA/PmrB system is well understood, those of the CpxR/CpxA system are more complex because they can be induced by various stress conditions, which, in turn, expresses a variety of phenotypes. Here, we highlight another aspect of the CpxR/CpxA system; mutations in degP and yqjA genes, which are under the control of the system, exhibit an iron sensitive phenotype in the mutant background defective in the PmrA-dependent gene products that alter the pyrophosphate status of the lipid A moiety of lipopolysaccharide in Salmonella enterica. Therefore, after the PmrA/PmrB-mediated Fe3+-dependent control of the pyrophosphate status on the cell surface, the CpxR/CpxA system is one of the second layers of envelope stress response that allows adaptation to high Fe3+ conditions in this bacterium.

In vivo cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in Salmonella enterica.

Recombineering has been used to facilitate the development of in vivo cloning methods. However, the method relies heavily on PCR, which still generates a much higher error rate than DNA replication in vivo, even when amplifying large DNA inserts. Here, a precise technique is reported in Salmonella enterica that enables the cloning of up to at least 19 kb target chromosomal DNA segments that had been marked by FRTs, which were derived from two consecutive lambda Red-mediated recombination events. P22 phage was utilized to transduce the target DNA segments from donor strains to recipient strains harboring a derivative of bacterial artificial chromosome (BAC) containing a FRT and a plasmid expressing Flp recombinase. This method was successful in cloning a gene cluster responsible for lipopolysaccharide (LPS) modifications that confer polymyxin B resistance and in complementing its mutant. Further optimized procedures should be widely applicable because large insert fragments are precise clones of the wild-type genome.

Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii.

UNLABELLED: Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii IMPORTANCE: A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.

Improved Lung Cancer Detection in Cardiovascular Outpatients by the Pulmonologist-based Interpretation of Chest Radiographs.

OBJECTIVE: Pulmonologists and cardiologists view chest radiographs differently. Lung cancer may therefore go undetected in patients referred to cardiovascular departments. We aimed to determine the clinical benefit of the additional interpretation of chest radiographs by pulmonologists in study involving cardiovascular outpatients. METHODS: A retrospective review of chest radiographs of outpatients attending a Japanese cardiovascular hospital between April 2000 and March 2010 was conducted. Lung cancer patients were categorized into 3 groups: group C, patients in whom tumors were detected by a cardiologist at the first visit; group P, patients in whom tumors were detected by the additional interpretation of a chest radiographs by a pulmonologist after a lesion was missed by a cardiologist; and group H, patients from an earlier period in which chest radiographs were only examined by a cardiologist. RESULTS: Cardiologists detected 9 cases of lung cancer in groups C and H from 2,430 and 2,288 radiographs, respectively. Pulmonologists detected 10 cases of lung cancer (group P) and 3 other malignancies that were previously undetected, giving a miss rate of 52.6% for the cardiologists. Tumor diameters were significantly smaller in group P than in group C or H. Furthermore, a significantly higher number of the tumors in group P were of an early stage and resectable, with more superposing structures than in groups C or H. CONCLUSION: The additional pulmonologist-based interpretations significantly increased the detection rate of operable tumors that mostly corresponded to the early T1 stage; this serves offers a potential clinical benefit in reducing the period of time from patient presentation to the diagnosis of lung cancer.

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella.

BACKGROUND: Bacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined. RESULTS: Here, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation. CONCLUSIONS: We identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system.

Reciprocal control between a bacterium's regulatory system and the modification status of its lipopolysaccharide.

Gram-negative bacteria often modify their lipopolysaccharide (LPS), thereby increasing resistance to antimicrobial agents and avoidance of the host immune system. However, it is unclear how bacteria adjust the levels and activities of LPS-modifying enzymes in response to the modification status of their LPS. We now address this question by investigating the major regulator of LPS modifications in Salmonella enterica. We report that the PmrA/PmrB system controls expression of a membrane peptide that inhibits the activity of LpxT, an enzyme responsible for increasing the LPS negative charge. LpxT's inhibition and the PmrA-dependent incorporation of positively charged L-4-aminoarabinose into the LPS decrease Fe(3+) binding to the bacterial cell. Because Fe(3+) is an activating ligand for the sensor PmrB, transcription of PmrA-dependent LPS-modifying genes is reduced. This mechanism enables bacteria to sense their cell surface by its effect on the availability of an inducing signal for the system regulating cell-surface modifications.

Gastric metastasis from a primary renal leiomyosarcoma.

Primary leiomyosarcoma of the kidney is rare. Here we report a case of metastasis of this tumor to the stomach. A 73-year-old man visited our hospital suffering from general weakness and intermittent tarry stools. He had undergone right nephrectomy for renal leiomyosarcoma 2 years previously. There had been no local recurrence or distant metastasis in the 2-year follow-up period. Endoscopy revealed two submucosal tumors in the stomach. These tumors were diagnosed histologically as leiomyosarcoma and distal gastrectomy was performed. Subsequent histochemical staining confirmed the diagnosis of gastric metastasis from renal leiomyosarcoma. The patient died due to metastases to the liver and bone 9 months after the operation. To the best of our knowledge, this is the first report of gastric metastasis from primary renal leiomyosarcoma.

Transdiaphragmatic intercostal hernia following blunt trauma.

We report a rare case of traumatic transdiaphragmatic intercostal hernia (TDIH) in an 85-year-old women who was transported to our hospital by ambulance after blunt trauma caused by the involvement in a motor vehicle accident. Chest and abdominal computed tomography (CT) revealed a left diaphragmatic rupture with a chest wall hernia involving loops of small intestine. An emergency operation was performed and led to a diagnosis of TDIH. Surgery via a thoracoabdominal incision in the left side was performed to reduce the hernia contents, and the diaphragmatic and intercostal defects were reconstructed by direct suture. CT scans were very helpful in detecting TDIH in this case.

Intrinsic negative feedback governs activation surge in two-component regulatory systems.

PhoP and PhoQ comprise a two-component system in the bacterium Salmonella enterica. PhoQ is the sensor kinase/phosphatase that modifies the phosphorylation state of the regulator PhoP in response to stimuli. The amount of phosphorylated PhoP surges after activation, then declines to reach a steady-state level. We now recapitulate this surge in vitro by incubating PhoP and PhoQ with ATP and ADP. Mathematical modeling identified PhoQ's affinity for ADP as the key parameter dictating phosphorylated PhoP levels, as ADP promotes PhoQ's phosphatase activity toward phosphorylated PhoP. The lid covering the nucleotide-binding pocket of PhoQ governs the kinase to phosphatase switch because a lid mutation that decreased ADP binding compromised PhoQ's phosphatase activity in vitro and resulted in sustained expression of PhoP-dependent mRNAs in vivo. This feedback mechanism may curtail futile ATP consumption because ADP not only stimulates PhoQ's phosphatase activity but also inhibits ATP binding necessary for the kinase reaction.

Laparoscopic splenectomy for idiopathic thrombocytopenic purpura in a woman with situs inversus: Report of a Case.

A 51-year-old woman with previously diagnosed situs inversus (SI) totalis was admitted to our hospital with nasal bleeding. Blood tests showed severe thrombocytopenia, with a platelet count of 1.9 × 104/mm(3), and idiopathic thrombocytopenic purpura (ITP) was diagnosed. She was refractory to medical treatment, so we performed laparoscopic splenectomy (LS). The mirrorimage anatomy made the procedure difficult for the right-handed surgeon, so we modified the technique to enable it to be performed via an anterior approach through the subxiphoid area using the right hand, with an ultrasonic dissecting device and an endostapling device. The operation was performed safely, the patient recovered uneventfully, and her platelet count normalized. To our knowledge, there has been no other report of LS in a patient with SI; however, it can be performed safely with careful management.

The PhoQ/PhoP regulatory network of Salmonella enterica.

The PhoQ/PhoP two-component regulatory system is a major regulator of virulence in the enteric pathogen Salmonella enterica serovar Typhimurium. It also controls the adaptation to low Mg2+ environments by governing the expression and/or activity of Mg2+ transporters and of enzymes modifying the Mg2+-binding sites on the bacterial cell surface. The regulator PhoP modifies expression of approximately 3% of the Salmonella genes in response to the periplasmic Mg2+ concentration detected by the PhoQ protein. Genes that are directly controlled by the PhoP protein often differ in their promoter structures, resulting in distinct expression levels and kinetics in response to the low Mg2+ inducing signal. PhoP regulates a large number of genes indirectly: via other transcription factors and two-component systems that form a panoply of regulatory architectures including transcriptional cascades, feedforward loops and the use of connector proteins that modify the activity of response regulators. These architectures confer distinct expression properties that may be important contributors to Salmonella's lifestyle.

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli.

Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.

A connector of two-component regulatory systems promotes signal amplification and persistence of expression.

Organisms rely on a variety of regulatory architectures to control gene transcription. Whereas the functional characteristics of particular architectures are well understood, the properties of newly discovered regulatory designs cannot be easily predicted. One emerging design depends on small proteins that connect two-component regulatory systems, which constitute the dominant form of bacterial signal transduction. These connectors enable one system to respond to the signal perceived by a different system. To understand the functional properties of such connector-mediated architectures, we investigated the pathway controlled by the PhoP-dependent connector protein PmrD of Salmonella enterica and contrasted it to the circuit in which genes are regulated directly by the transcription factor PhoP. The PmrD-mediated pathway displayed both signal amplification and persistence of expression when compared with the direct pathway. Mathematical modeling of the two pathways allowed us to identify critical factors responsible for signal amplification.

Rapid and simple detection of hot spot point mutations of epidermal growth factor receptor, BRAF, and NRAS in cancers using the loop-hybrid mobility shift assay.

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed.

Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica.

Genetic and genomic approaches have been successfully used to assign genes to distinct regulatory networks. However, the present challenge of distinguishing differentially regulated genes within a network is particularly hard because members of a given network tend to have similar regulatory features. We have addressed this challenge by developing a method, termed Gene Promoter Scan, that discriminates coregulated promoters by simultaneously considering both multiple cis promoter features and gene expression. Here, we apply this method to probe the regulatory networks governed by the PhoP/PhoQ two-component system in the enteric bacteria Escherichia coli and Salmonella enterica. Our analysis uncovered members of the PhoP regulon and interactions with other regulatory systems that were not discovered in previous approaches. The predictions made by Gene Promoter Scan were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, regulates acid resistance determinants, and is a central element in a highly connected network.