bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Hydraulic System for Mini Excavators Without Electronic Controls
Authors: Sugimura Ken , Murrenhoff Hubertus
Published: November 2016

The target application of this study is hydraulic excavators, which are one of the most common machines found at construction sites across the world. Road constructions and improvements, laying operation of cables or pipes and building can be seen in urban areas and digging and dumping operations of natural resource are done in country regions. For the construction site in urban areas, mini hydraulic excavators with operating weights up to 6 tons are often used and they make up more than 60% of the total hydraulic excavators market [1].

In recent years, a number of new system architectures for mobile hydraulic systems have been proposed. Examples of such improved architectures are displacement control, transformer systems and valve controlled systems with multiple pressure rails. For these systems, electronic controls are always used. Although these new methods are promising, they cannot be applied to mini-excavators, because today’s mini-hydraulic excavators do not use electronic controls as this would increase costs and make the system complex. Therefore, the goal of this study is to propose a fully hydro-mechanical valve controlled constant pressure system, which can be applied to mini-excavators in the future.

This paper begins by introducing the details of this novel hydraulic system and shows its advantages. Using a simulation model of an 18 ton excavator, it is confirmed that the novel system functions well and the energy efficiency is compared to a conventional Load Sensing system. The simulation results show that the novel system can save 22% and 24% of fuel in leveling and 90° dig-dump cycles respectively.
Pump Efficiency Parameter Estimation
Authors: Shinn Tyler , Carpenter Richard , Fales Roger C.
Published: November 2016

Hydraulic pump health monitoring can give early notice of a catastrophic failure occurring within the pump, saving time and money on repairs. This work focuses on developing a system to monitor an axial piston pump’s volumetric efficiency, using state and parameter estimation techniques. A high order, nonlinear model has been utilized for the axial piston pump. Pressure measurements of the pump are used for a linear Kalman filter (KF) as well as an extended Kalman filter (EKF) to estimate the remaining states of pump model. Volumetric efficiency losses are tracked by the filters via estimation of two flow loss coefficients, low Reynolds and high Reynolds flow loss, which are allowed to vary within the model to track the changes. In a separate analysis, a third parameter, a disturbance torque, was applied to the load and its estimation in a similar process to the flow loss coefficients.

Both filters are able to estimate a single flow loss or load. However, the KF was unable to distinguish between two flow losses. The EKF is able to distinguish between low and high Reynolds number flows since it takes into account the nonlinearities in the system including the flow loss characteristics. The EKF shows promise in being able to estimate both flow losses and a load disturbance simultaneously. Both types of filters are found to have fast run times suggesting that the filters could be implemented using typical microcontroller hardware found on industrial and mobile hydraulic machinery.
The Design, Analysis and Testing of a Compact Electrohydrostatic Powered Ankle Prosthesis
Authors: Yu Tian , Plummer Andrew , Iravani Pejman , Bhatti Jawaad , Zahedi Saeed , Moser David
Published: November 2016

This paper presents a prototype powered ankle prosthesis which can operate passively in most of the gait cycle and provide assistance for toe push-off and subsequent foot dorsiflexion. The use of electrohydrostatic actuation (EHA) gives the ability to switch quickly and smoothly between passive and active modes. In this new powered ankle prosthesis, the motor-pump unit is integrated with the ankle joint and the battery and controller are held in a backpack. A 100W brushless DC motor is used driving a 0.45cc/rev gear pump. The motor runs in hydraulic fluid, pressurised to 60bar, avoiding the need for a pump shaft seal and a refeeding circuit for external leakage. A simulation model was developed to help analyse the performance characteristics of the EHA. The simulation results are compared with laboratory-based experiment results to validate the model. The compact prototype is suitable for prolonged testing with amputees, and an example set of results from amputee testing is presented.
Methodical Loss Reduction in Load Sensing Systems Based on Measurements
Authors: Hießl Arnold , Scheidl Rudolf
Published: November 2016

Energy efficiency improvements are forced by steadily increasing general performance and cost saving requirements, and for mobile machines mostly by stricter governmental emission laws. Such improvements can be realized by new system architectures, like hybrids or energy recovery systems, but also by optimizing existing systems. This publication discusses the reduction of systematic losses for an existing hydraulic Load Sensing System (LSS) used in mobile working machines, especially in compact excavators. Energy losses in a LSS are proportional to the pressure difference between pump and actuator in each section. These systematic losses are investigated and can be reduced by actuator adaptation or by splitting non-correlating sections. Energy losses along the hydraulic circuit, such as pump losses hydraulic line losses and actuator losses, which are affected by these adaptations indirectly, are neglected.

The investigations are founded on measurements of a 5 ton compact excavator and their systematic evaluation. The actuator adaptation can be realised by changing the excavator’s geometry and/or hydraulic specifications (cylinder areas, displacement volume). The focus of this paper is limited to the hydraulic domain. Mathematical models and operation scenarios verified by measurements were taken as the basis to find optimum system parameter configurations by mathematical optimization, employing evolutionary algorithm. This included also different groupings of LSS circuits. Boom, stick, bucket and swing were taken into account and results are shown for a one, two and three pump LSS. Considering the introduced methods an effective way for reducing systematic losses up to 40% is shown in this exemplary case.
Characteristics Prediction of Vane Pump With Rich Gases Containing Hydraulic Oil
Authors: Nakamura Yoshinari , Yakabe Sinji , Suzuki Kazunari , Watanabe Hirohito , Tsukiji Tetsuhiro
Published: November 2016

In recent years, fuel consumption of vehicles has been remarkably improved by some cutting edge technologies (e.g., hybrid electric vehicles, non-idling systems). In-vehicle equipment is also required to improve efficiency. The vane pumps used to control power steering and transmissions must demonstrate maximum performance, even under high temperature conditions and with rich gases contained in hydraulic oil. Precise recognition of the dynamics of pump inner flow can help in the design and development of vane pumps.

In this study, the 3-dimensional CFD is adopted to analyze the characteristics of inner flow dynamics of vane pumps. Since the gases contained in hydraulic oil influence the characteristics of vane pumps, the two-phase flow model with cavitation is assumed in particular. First, this CFD was verified by comparison with experimental results which were carried out at higher rotational speeds and with oil containing a high level of gas; the results showed the error of the suction flow rate could be as little as 5% or less. Internal pressure in the vane chambers was then studied to identify the relationship between the cavitation model and the contained gas ratio of the oil. The simulation results further clarified that the gas clouds deformed by pump rotation could restrict the flow of the suction ports.
Replacing a Constant Pressure Valve Controlled System With a Pump Controlled System
Authors: Hänninen Henri , Minav Tatiana , Pietola Matti
Published: November 2016

Traditionally, a typical hydraulic circuit utilized in stationary industrial applications is based on valve operated actuation. One realization of such a system is a constant pressure circuit employing a hydraulic accumulator as an energy reserve and pressure stabilizer. The pump is used to maintain the desired pressure level, for example by using a variable displacement pump that controls the displacement setting based on the pressure level.

The main benefit of this system architecture is its ability to produce high output powers with a very low response time. However, it is not the most energy efficient and in many cases, not the most space efficient solution. The efficiency of this system type is reduced mainly by the need to choke the pressure difference between the set system pressure and the actual pressure need in the actuator. By directly controlling the actuator via controlling the pump’s output flow with an electric servo motor, the throttling losses of the valve controlled system can be avoided. In addition, this enables the usage of closed circuits which in terms removes the need for a large reservoir.

In this study, the replacement of a valve controlled hydraulic system with a pump controlled system in an industrial stationary material handling machine is investigated. The machine’s work cycle consist of continuous consecutive lifting and lowering motions of one end of a platform pivoted at the opposite end. The study consist of designing the replacing circuit topology, of dimensioning the hydraulic components utilizing a created Simulink-based tool and of a simulation based analysis on the dynamic properties of the designed hydraulic system.
The Dynamics of Leakage in Bent Axis Units Without Timing Gear
Authors: Manhartsgruber Bernhard
Published: November 2016

Bent axis hydraulic pumps and motors are extremely popular due to their high efficiency and large speed range. A number of different concepts exist with respect to kinematic restraints on the cylinder barrel motion. Some manufacturers rely upon a timing gear for precise synchronization of the shaft and barrel speeds while other companies have successfully introduced bent axis units without such a mechanism. The paper analyses the dynamics of bent axis machines with tapered pistons driving the cylinder barrel. A rotation of the pistons inside the corresponding bores is proposed to result in changing cylinder chamber to case drain leakages. The reported phenomenon is shown to have a significant effect on the low frequency part of pressure and flow pulsations. In this way, frequency components far below the fundamental frequency associated with the shaft revolution are generated.
Dynamic Equivalence of Pneumatic and Electrical Boost Converters for Exhaust Gas Energy Reclamation
Authors: Gibson Tyler J. , Barth Eric J.
Published: November 2016

Significant usable energy is discarded as exhaust gas in most pneumatic processes. The ability to recycle this energy could lead to significant improvements in system efficiency. This paper presents a method of dynamically converting the exhaust gas energy of pneumatic systems to a higher pressure so that it may be reintroduced to the pressure supply and reused, boosting energy efficiency of industrial pneumatic systems. This is the pneumatic equivalent of a boost converter, an electrical system that supplies a greater voltage to a load than the power source can supply. Each component of the electrical system can be analogized to an equivalent pneumatic component. The most apparent of these comparisons is the method of storing and transforming energy. In the electrical system, the energy is stored in an inductor which is charged in a closed loop. In the pneumatic system, energy can be stored as momentum. When this stored energy is discharged, a spike in voltage or pressure will be observed in the electrical or pneumatic system, respectively. Similarly, every component of the electrical boost converter can be linked to a pneumatic counterpart. With these relationships fully understood, a device to perform the pneumatic boost conversion is modeled. Successful realization of this result will confirm the analogy between the electrical and pneumatic systems, which will allow for the development of more complex pneumatic systems based on various well understood electrical converters. This paper presents simulations of both electrical and pneumatic boost converters. Insights regarding the energy conversion and its efficiency are drawn from the pneumatic model as well as from the dynamically similar electrical model.
Carbon emissions from land-use change and management in China between 1990 and 2010
Authors: Lai Li , Huang Xianjin , Yang Hong , Chuai Xiaowei , Zhang Mei , Zhong Taiyang , Chen Zhigang , Chen Yi , Wang Xiao , Thompson Julian R.
Published: November 2016

Overall land-use change and management have contributed about 1.45 Pg of carbon to the total carbon released in China from 1990 to 2010.
Chemical delivery array with millisecond neurotransmitter release
Authors: Jonsson Amanda , Sjöström Theresia Arbring , Tybrandt Klas , Berggren Magnus , Simon Daniel T.
Published: November 2016

Addressable organic electronic neurotransmitter delivery array with switching ~50 ms, approaching synaptic signaling speed.
Ambitious web fundraising startup fails to meet big goals
Authors: Harris Mark
Published: November 2016

Hundreds of scientists paid fees to have their work featured on Benefunder, but very few received donations
‘Four-legged snake’ may be ancient lizard instead
Authors: Gramling Carolyn
Published: November 2016

Controversy swirls around fossil, which is no longer accessible
Mice made easy
Authors: Cohen Jon
Published: November 2016

The genome-editing tool CRISPR upends the vital business of creating mutant mice
A reporter does CRISPR
Authors: Cohen Jon
Published: November 2016

Can “any idiot” succeed at using this powerful new genome-editing tool?
Pot and pain
Authors: Miller Greg
Published: November 2016

Hints are emerging that cannabis could be an alternative to opioid painkillers
Primed for pain
Authors: Servick Kelly
Published: November 2016

Do opioids make chronic users more sensitive to pain?
Tips for battling billion-dollar beetles
Authors: Tabashnik Bruce E.
Published: November 2016

Tiny insecticidal proteins (Tips) kill pests that evolved resistance to engineered corn
Human Parvoviruses
Authors: Qiu Jianming , Söderlund-Venermo Maria , Young Neal S.
Published: November 2016

Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large
Parvoviridae
family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable
in vitro
, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology.
O-antigen variation in Salmonella spp.: rfb gene clusters of three strains
Authors: Verma N K , Quigley N B , Reeves P R
Published: November 2016

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side-chain sugar residue. These genes encoding O-antigen biosynthesis are clustered in the rfb operon. We report here the molecular cloning and analysis of the rfb operons of Salmonella paratyphi A (serogroup A) and S. typhi (serogroup D). The regions of DNA nonhomology between the rfb operons of these serogroup A, B, and D representatives are identified, and the evolutionary derivation of serogroup A from a serogroup D progenitor is discussed.
Rhizobium japonicum USDA 191 has two nodD genes that differ in primary structure and function
Authors: Appelbaum E R , Thompson D V , Idler K , Chartrain N
Published: November 2016

Several Rhizobium genes (designated nod genes) are involved in early steps in nodule formation. Here we present the results of DNA sequence and functional analysis of two nodD genes from the symbiotic plasmid of USDA 191, a fast-growing strain that forms nitrogen-fixing nodules on soybeans. Both genes encoded full-length nodD-related polypeptides, which were 69% homologous to each other. One of these genes, nodD1, complemented a Rhizobium trifolii nodD::Tn5 mutant for clover nodulation; the other gene, nodD2, did not. The nodD1 coding region was preceded by a conserved DNA sequence previously noted in other rhizobia, but no such sequence was found in front of nodD2. Plants inoculated with a nodD1 insertion mutant appeared to be nitrogen starved and had a greatly reduced nodule number. Plants inoculated with a nodD2 mutant had a partially nitrogen-starved appearance and normal nodule number, were slightly delayed in nodule formation, and formed nodules that contained reduced levels of nodulin-35 and had fewer bacteroids per infected plant cell. Thus, both of these genes are involved in symbiosis. USDA 191 carrying extra copies of nodD2 on a plasmid vector had an altered colony morphology that suggested inhibition of exopolysaccharide synthesis. The predicted gene products of nodD1 and nodD2 both showed homology to LysR, an E. coli regulatory protein. We conclude that nodD1 probably has the same function as nodD in temperate rhizobia, namely, activation of nodABC transcription in the presence of plant signals. nodD2 may be involved in regulation of exopolysaccharide synthetic genes.
Identification of a Staphylococcus aureus transposon (Tn4291) that carries the methicillin resistance gene(s)
Authors: Trees D L , Iandolo J J
Published: November 2016

We isolated a transposon (Tn4291) that carries the resistance gene(s) for methicillin in a secondary insertion site on the penicillinase plasmid pI524. Transposition of Tn4291 into pI524 occurred during the transduction of the tetracycline resistance plasmid pSN1 from a methicillin-resistant donor into a recipient that carried the mec allele in the primary site on the chromosome. Insertion of Tn4291 caused extensive rearrangement of pI524 and resulted in the formation of a 27.9-kilobase-pair plasmid (pIT103) which coded for resistance to methicillin and cadmium, but not penicillin. Although resistance to methicillin and cadmium were always linked, Tn4291 was stably maintained only in the presence of a chromosomal mec allele, while in its absence the plasmid was unstable and transposition to the primary site occurred. Subsequently, a 20.1-kilobase-pair plasmid, pIT203, was formed which retained cadmium resistance and regained the ability to express beta-lactamase activity.
Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound
Authors: Sadowsky M J , Olson E R , Foster V E , Kosslak R M , Verma D P
Published: November 2016

Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.
Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit
Authors: Miki J , Maeda M , Futai M
Published: November 2016

A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified. This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C. When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity. Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes. The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C. These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S. D. Dunn and M. Futai, J. Biol. Chem. 255:113-118, 1980).
Gene organization and structure of the Streptomyces lividans gal operon
Authors: Adams C W , Fornwald J A , Schmidt F J , Rosenberg M , Brawner M E
Published: November 2016

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.
Regulation of nitrogenase synthesis in histidine auxotrophs of Klebsiella pneumoniae with altered levels of adenylate nucleotides
Authors: Stougaard J , Kennedy C
Published: November 2016

A histidine auxotrophic (hisA) mutant of Klebsiella pneumoniae is phenotypically Nif- when grown with 20 micrograms of histidine ml-1 but Nif+ when supplied with histidine at 100 micrograms ml-1. Reversion to Nif+ at 20 micrograms of histidine ml-1 occurs phenotypically by the addition of 2-thiazolyl-DL-alanine or genetically by mutation in hisG; 2-thiazolyl-DL-alanine inhibits and hisG encodes phosphoribosyl phosphotransferase, the first enzyme of the histidine biosynthetic pathway which consumes ATP. Physiological studies of the hisA mutant JS85 showed that after removal of NH4+ from a culture of the mutant grown with 20 micrograms of histidine ml-1, synthesis of nitrogenase polypeptides occurred at a rate similar to that in the wild type for about 3 h and acetylene reduction activity reached about 10% of the fully derepressed wild-type level. Shortly thereafter the concentration of intracellular adenylates decreased; in particular, ATP fell to about 10% of normal levels. Also, nitrogenase proteins (nifHDK products) and the nifJ gene product stopped being synthesized. These effects were not due to impairment of growth or protein synthesis by histidine starvation. Inhibition of phosphoribosyl phosphotransferase with 2-thiazolyl-DL-alanine restored nitrogenase activity and synthesis, indicating that the effect of the hisA mutation on nif expression was probably a consequence of lowered energy resources that occurred during anaerobic N starvation. The loss of ATP was not associated with nitrogenase synthesis or activity, since hisA nifA and hisA nifH double mutants underwent a loss of ATP in derepressing conditions. Transcription from the nifL, nifN, and nifH promoters was examined in hisA strains with Mu d(Ap lac) fusions in these nif genes. Transcription was not significantly influenced under conditions where adenylates were decreased in concentration. Also nif mRNA apparently accumulated in cultures unable to synthesize nitrogenase, suggesting that translational control of nif gene product synthesis occurs under unfavorable energetic conditions.
Molecular cloning of the wild-type phoM operon in Escherichia coli K-12
Authors: Wanner B L , Wilmes M R , Hunter E
Published: November 2016

A metastable bacterial alkaline phosphatase (Bap) phenotype is seen in phoR mutants, which alternately express a Bap-constitutive or -negative phenotype. The alteration is affected by mutations in the phoM region near 0 min. By molecular cloning of the wild-type phoM operon onto a multicopy plasmid and recombining onto the plasmid the pho-510 mutation that abolishes variation, the phoM operon, rather than some nearby gene, was shown to control variation. Complementation tests indicated that the wild-type phoM allele is dominant to the pho-510 mutation when both are in single copy, but whichever allele is present in higher copy appears as dominant when multicopy plasmids are examined. The alternating phenotypic variation of BAP synthesis was not seen in phoR+ cells with multicopy wild-type phoM plasmids, thus showing that the variation is associated with phoM-dependent Bap expression. The alternation acted at the level of phoA transcription; it was also recA independent. BAP clonal variation is phenotypically similar to Salmonella phase variation, which is controlled by a DNA rearrangement. No evidence was found for a DNA change near the phoM operon that might be responsible for the variable Bap phenotype.
Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides
Authors: Davis J , Donohue T J , Kaplan S
Published: November 2016

A Puf- strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf- phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS+ phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.
DNA sequence of the D-serine deaminase activator gene dsdC
Authors: Palchaudhuri S , Patel V , McFall E
Published: November 2016

We have determined the DNA sequence of dsdC, the gene that encodes the D-serine deaminase activator protein of Escherichia coli K-12. The sequence contains a single open reading frame that terminates in a UGA codon. One the basis of the size of the protein, 33 kilodaltons, and the amino acid sequence encoded by the open reading frame, we identified a likely translation initiation codon 731 base pairs upstream of the translation initiation codon for the divergently transcribed D-serine deaminase gene. There is a broad range of codon usage, not surprising in view of the weak expression of the gene. The N-terminal two-thirds of the activator is arginine-lysine rich and quite polar; the remainder is more neutral. The segment of the protein that seems most likely to have potential to form the helix-turn-helix structure characteristic of DNA-regulatory proteins is located near the end of the polar region. The protein contains a region with significant homology to lambda attB.
Nucleotide sequence of the type A staphylococcal enterotoxin gene
Authors: Betley M J , Mekalanos J J
Published: November 2016

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.
Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks
Authors: Baldwin W W , Sheu M J , Bankston P W , Woldringh C L
Published: November 2016

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.
Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees C
Authors: Bryan S K , Moses R E
Published: November 2016

Escherichia coli cells with the pcbA1 allele and temperature-sensitive DNA polymerase III mutations survived at a restrictive temperature if DNA polymerase I activity was present. The Klenow fragment of DNA polymerase I was capable of supporting cell survival as well.
Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes
Authors: Angov E , Brusilow W S
Published: November 2016

In-frame fusions to lacZ were constructed in two adjacent genes of the unc operon of Escherichia coli, uncA and uncG, which code for the alpha and gamma subunits of the proton-translocating ATPase. After each fusion was moved into the E. coli chromosome, measurement of beta-galactosidase activities from single-copy genes showed that uncA was expressed significantly better in vivo than was uncG, but the relative expression dependent on the chromosomal location of each fusion and the presence or absence of other unc genes.
Rapid identification of bacterial genes that are lethal when cloned on multicopy plasmids
Authors: Berg C M , Liu L , Wang B , Wang M D
Published: November 2016

A procedure to identify genes that are lethal when cloned on multicopy plasmids was developed. It depends on the ability of mini-Mu plasmid elements to be used for both in vivo cloning and generalized transduction of enterobacterial genes. The feasibility of this procedure was demonstrated by using the tetA gene of Tn10, which is lethal when in multiple copies in the presence of 25 micrograms of tetracycline per ml.
Cloning and sequencing of a gene encoding an aminoglycoside 6'-N-acetyltransferase from an R factor of Citrobacter diversus
Authors: Tenover F C , Filpula D , Phillips K L , Plorde J J
Published: November 2016

The aacA1 gene, which encodes a 6'-N-acetyltransferase [AAC(6')-I] mediating resistance to kanamycin, tobramycin, and amikacin, was cloned from the Citrobacter diversus R plasmid pBWH100 into the Escherichia coli vector pBR322. The complete nucleotide sequence of the gene and flanking regions was determined. A protein of approximately 21 kilodaltons was identified when the chimeric plasmid encoding the aacA1 gene was introduced into E. coli maxicells. This value is consistent with the size predicted for a protein translated from the open reading frame of the gene.
Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant
Authors: Resnick D , Nelson D R
Published: November 2016

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.
Synthesis of bacilysin by Bacillus subtilis branches from prephenate of the aromatic amino acid pathway
Authors: Hilton M D , Alaeddinoglu N G , Demain A L
Published: November 2016

Previously characterized mutants of Bacillus subtilis 168 were analyzed for the accumulation of the antibiotic bacilysin in culture broths and for the presence of bacilysin synthetase in cell extracts. All aro mutants tested were deficient in bacilysin biosynthesis but had synthetase activity. Mutants with lesions in tyrA and pheA produced normal levels of bacilysin, suggesting that prephenate is the primary metabolic precursor of bacilysin.
Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion
Authors: Duplay P , Hofnung M
Published: November 2016

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm.
Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC7942
Authors: Reddy K J , Bullerjahn G S , Sherman D M , Sherman L A
Published: November 2016

We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.
Nucleotide sequence of the xth gene of Escherichia coli K-12
Authors: Saporito S M , Smith-White B J , Cunningham R P
Published: November 2016

The xth gene of Escherichia coli K-12, which encodes exonuclease III, has been sequenced. Exonuclease III from a cloned copy of the E. coli K-12 gene has been purified and characterized. The molecular weight (30,921), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme. The xth promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence reveals that a region of dyad symmetry which could form a hairpin stem-loop structure in RNA characteristic of a rho-dependent terminator lies immediately downstream from the xth gene.
Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum
Authors: Grépinet O , Chebrou M C , Béguin P
Published: November 2016

The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon.
DNA polymorphism in strains of the genus Brucella
Authors: Allardet-Servent A , Bourg G , Ramuz M , Pages M , Bellis M , Roizes G
Published: November 2016

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.
Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides
Authors: Oelze J
Published: November 2016

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.
Transcriptional analysis of the major surface array gene of Caulobacter crescentus
Authors: Fisher J A , Smit J , Agabian N
Published: November 2016

The major component of the paracrystalline surface array of Caulobacter crescentus CB15 and one of the most abundant cellular proteins is a protein designated 130K. We have determined the DNA sequence of the 5' portion of the 130K gene, including the N-terminal one-third of the protein coding region, and analyzed the transcription of the gene. The site of transcription initiation was determined by S1 mapping of Caulobacter RNA. Although the DNA sequence upstream from the transcription start site showed significant homology to the consensus promoter sequences of Escherichia coli, S1 analysis of RNA from E. coli carrying the 130K gene on a plasmid indicated that the 130K promoter was not transcribed by E. coli RNA polymerase in vivo. Quantitative S1 analysis of RNA isolated from synchronously growing Caulobacter cells suggested that this promoter was not under developmental regulation; the amount of 130K transcript varied no more than 1.5-fold during the cell cycle. The length of the 130K mRNA was determined to be 3.3 kilobases by Northern (RNA blot) analysis, indicating that the 130K mRNA is not part of a polycistron. The amino acid sequence predicted from the DNA sequence agreed well with the N-terminal amino acid sequence determined by sequencing of the 130K protein. The 130K protein appears to be synthesized without an N-terminal leader sequence, but the N-terminal 20 amino acids are relatively hydrophobic and may function like a signal sequence during transmembrane translocation.
Structure and comparative analysis of the genes encoding component C of methyl coenzyme M reductase in the extremely thermophilic archaebacterium Methanothermus fervidus
Authors: Weil C F , Cram D S , Sherf B A , Reeve J N
Published: November 2016

A 6-kilobase-pair (kbp) region of the genome of the extremely thermophilic arachaebacterium Methanothermus fervidus which encodes the alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase was cloned and sequenced. Genes encoding the beta (mcrB) and gamma (mcrG) subunits were separated by two open reading frames (designated mcrC and mcrD) which encode unknown gene products. The M. fervidus genes were preceded by ribosome-binding sites, separated by short A + T-rich intergenic regions, contained unexpectedly few NNC codons, and exhibited inflexible codon usage at some locations. Sites of transcription initiation and termination flanking the mcrBDCGA cluster of genes in M. fervidus were identified. The sequences of the genes, the encoded polypeptides, and transcription regulatory signals in M. fervidus were compared with the functionally equivalent sequences from two mesophilic methanogens (Methanococcus vannielii and Methanosarcina barkeri) and from a moderate thermophile (Methanobacterium thermoautotrophicum Marburg). The amino acid sequences of the polypeptides encoded by the mcrBCGA genes in the two thermophiles were approximately 80% identical, whereas all other pairs of these gene products contained between 50 and 60% identical amino acid residues. The mcrD gene products have diverged more than the products of the other mcr genes. Identification of highly conserved regions within mcrA and mcrB suggested oligonucleotide sequences which might be developed as hybridization probes which could be used for identifying and quantifying all methanogens.