bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of frequency cueing on the perceptual segregation of simultaneous tones: Bottom-up and top-down contributions
Authors: Shen Yi
Published: November 2016

Listeners were presented with two simultaneous tones of different frequencies (more than one octave apart) and asked to identify the tone that was amplitude-modulated while a tonal precursor was presented to cue the frequency of the lower frequency tone. Performance thresholds were estimated based on the duration of the tone-pair. In Exp. I the duration of the precursor varied from 100 to 400 ms and the inter-stimulus interval (ISI) between the precursor and the tone-pair varied from 0 to 1 s. The presence of the precursor facilitated segregation. As the ISI increased, the facilitation effect of the precursor increased for the precursor durations of 100 and 200 ms, but not for the 400-ms precursor duration. When the precursor was presented to the contralateral ear relative to the tone-pair in Exp. II, no significant change to the precursor effect was observed. These observations contradict the predictions of the model based solely on bottom-up processing, suggesting the likely involvement of top-down processes.
Optimization of Synergistic Combination Regimens against Carbapenem- and Aminoglycoside-Resistant Clinical Pseudomonas aeruginosa Isolates via Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling
Authors: Yadav Rajbharan , Bulitta Jürgen B. , Nation Roger L. , Landersdorfer Cornelia B.
Published: November 2016

Optimizing antibiotic combinations is promising to combat multidrug-resistant
Pseudomonas aeruginosa
. This study aimed to systematically evaluate synergistic bacterial killing and prevention of resistance by carbapenem and aminoglycoside combinations and to rationally optimize combination dosage regimens via a mechanism-based mathematical model (MBM). We studied monotherapies and combinations of imipenem with tobramycin or amikacin against three difficult-to-treat double-resistant clinical
P. aeruginosa
isolates. Viable-count profiles of total and resistant populations were quantified in 48-h static-concentration time-kill studies (inoculum, 10
7.5
CFU/ml). We rationally optimized combination dosage regimens via MBM and Monte Carlo simulations against isolate FADDI-PA088 (MIC of imipenem [MIC
imipenem
] of 16 mg/liter and MIC
tobramycin
of 32 mg/liter, i.e., both 98th percentiles according to the EUCAST database). Against this isolate, imipenem (1.5× MIC) combined with 1 to 2 mg/liter tobramycin (MIC, 32 mg/liter) or amikacin (MIC, 4 mg/liter) yielded ≥2-log
10
more killing than the most active monotherapy at 48 h and prevented resistance. For all three strains, synergistic killing without resistance was achieved by ≥0.88× MIC
imipenem
in combination with a median of 0.75× MIC
tobramycin
(range, 0.032× to 2.0× MIC
tobramycin
) or 0.50× MIC
amikacin
(range, 0.25× to 0.50× MIC
amikacin
). The MBM indicated that aminoglycosides significantly enhanced the imipenem target site concentration up to 3-fold; achieving 50% of this synergistic effect required aminoglycoside concentrations of 1.34 mg/liter (if the aminoglycoside MIC was 4 mg/liter) and 4.88 mg/liter (for MICs of 8 to 32 mg/liter). An optimized combination regimen (continuous infusion of imipenem at 5 g/day plus a 0.5-h infusion with 7 mg/kg of body weight tobramycin) was predicted to achieve >2.0-log
10
killing and prevent regrowth at 48 h in 90.3% of patients (median bacterial killing, >4.0 log
10
CFU/ml) against double-resistant isolate FADDI-PA088 and therefore was highly promising.
Bacterial Replication Rate Modulation in Combination with Antimicrobial Therapy: Turning the Microbe against Itself
Authors: Ambrose Paul G. , VanScoy Brian , Conde Haley , McCauley Jennifer , Rubino Christopher M. , Bhavnani Sujata M.
Published: November 2016

A major clinical challenge for treating infectious diseases is the duration of antimicrobial therapy required to eradicate the pathogen. We hypothesized that modulation of the bacterial replication rate in the context of an antimicrobial exposure is coupled with the rate and extent of bactericidal effects. Herein we describe results from
in vitro
infection model (one compartment, 24-h model; hollow fiber, 10-day model) studies designed to probe the relationship between the bacterial replication rate and the rate and extent of bactericidal effects in the context of an effective antibiotic exposure. The bacterial replication rate was modulated by adjusting the sodium chloride concentration (0 to 8%) in the growth media (Mueller-Hinton II broth). The study drug selected was levofloxacin, and the challenge isolate was
Staphylococcus aureus
ATCC 29213 (levofloxacin MIC, 0.125 mg/liter). Within each
in vitro
infection model, human levofloxacin concentration-time profiles (half-life, 7 h) were simulated and the challenge isolate was subjected to an effective exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio], 65; administered as a single dose or daily for 10 days). Over the course of each study, samples were taken from each model for bacterial density determinations and drug concentration assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the 24-h one-compartment
in vitro
infection model studies, as the bacterial replication rate increased, so too did the rate (slope, 0 to 4 h) and extent (24-h CFU count per milliliter) of bacterial killing. In the 10-day hollow-fiber infection model studies, the times until a reduction of bacterial density to 1 × 10
2
CFU/ml occurred were 10 days in the media in which the challenge isolate grew slowly and approximately 2 days in the media in which the challenge isolate grew rapidly. Together, these data provide a proof of concept for new adjunctive therapeutic options with respect to the use of antimicrobial agents alone that reduce treatment durations. Such adjunctive therapies hold promise for marked reductions in the tonnage of antimicrobial agents administered to patient populations and selection pressure toward antimicrobial resistance.
Activity of Electrical Current in Experimental Propionibacterium acnes Foreign-Body Osteomyelitis
Authors: Schmidt-Malan Suzannah M. , Brinkman Cassandra L. , Greenwood-Quaintance Kerryl E. , Karau Melissa J. , Mandrekar Jayawant N. , Patel Robin
Published: November 2016

Foreign-body-associated infections are often difficult to treat, given that the associated microorganisms are in a biofilm state. Previously, we showed that a low-amperage direct electrical current (DC) reduces
Propionibacterium acnes
biofilms formed on implant-associated materials
in vitro
. In this study, low-amperage DC was compared to ceftriaxone treatment or no treatment in a novel rat femur model of foreign-body osteomyelitis. A platinum implant seeded with a
P. acnes
biofilm (10
7
CFU/cm
2
) and 10
9
CFU of planktonic
P. acnes
was placed in the femoral medullary cavity. One week later, rats were assigned to one of three treatment groups: no treatment, ceftriaxone treatment, or 200-μA-DC treatment. After 2 weeks of treatment, there were fewer bacteria in the bones of the ceftriaxone group (3.06 log
10
CFU/g of bone [
P
= 0.0209]) and the 200-μA-DC group (0.5 log
10
CFU/g [
P
= 0.0015]) than in those of the control group (6.58 log
10
CFU/g). The DC-exposed animals exhibited fewer bacteria than the ceftriaxone-treated animals (
P
= 0.0330). There were fewer bacteria on the implanted wires in the groups treated with ceftriaxone (0.1 log
10
CFU/cm
2
) or a 200-μA DC (0.1 log
10
CFU/cm
2
) than in the control group (2.53 log
10
CFU/cm
2
[
P
, 0.0003 for both comparisons]). Low-amperage DC may be useful for treating, or aiding in the treatment of, foreign-body infections caused by
P. acnes
.
Role of RNase Y in Clostridium perfringens mRNA Decay and Processing
Authors: Obana Nozomu , Nakamura Kouji , Nomura Nobuhiko
Published: November 2016

RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen
Clostridium perfringens
, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in
C. perfringens
and involved the
virR-virS
two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of
colA
(encoding a major toxin collagenase) and
pilA2
(encoding a major pilin component of the type IV pili). Posttranscriptional regulation of
colA
is known to be mediated by cleavage in the 5′ untranslated region (5′UTR), and we observe that RNase Y depletion diminishes
colA
5′UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of
pilA2
mRNA, which is thought to be important for host cell adherence and biofilm formation.

IMPORTANCE
RNases have important roles in RNA degradation and turnover in all organisms.
C. perfringens
is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding
C. perfringens
specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in
C. perfringens
results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of
C. perfringens
.
Effect of outer membrane permeability on chemotaxis in Escherichia coli
Authors: Ingham C , Buechner M , Adler J
Published: November 2016

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.
DNA sequence and translational product of a new nodulation-regulatory locus: syrM has sequence similarity to NodD proteins
Authors: Barnett M J , Long S R
Published: November 2016

Rhizobium meliloti nodulation (nod) genes are expressed when activated by trans-acting proteins in the NodD family. The nodD1 and nodD2 gene products activate nod promoters when cells are exposed to plant-synthesized signal molecules. Alternatively, the same nod promoters are activated by the nodD3 gene when nodD3 is carried in trans along with a closely linked global regulatory locus, syrM (symbiotic regulator) (J. T. Mulligan and S. R. Long, Genetics 122:7-18, 1989). In this article we report the nucleotide sequence of a 2.6-kilobase SphI fragment from R. meliloti SU47 containing syrM. Expression from this locus was confirmed by using in vitro transcription-translation assays. The open reading frame encoded a protein of either 33 or 36 kilodaltons whose sequence shows similarity to NodD regulatory proteins.
Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes
Authors: Boettcher K J , Ruby E G
Published: November 2016

Bioluminescent marine bacteria of the species Vibrio fischeri are the specific light organ symbionts of the sepiolid squid Euprymna scolopes. Although they share morphological and physiological characteristics with other strains of V. fischeri, when cultured away from the light organ association the E. scolopes symbionts depress their maximal luminescence over 1,000-fold. The primary cause of this reduced luminescence is the underproduction by these bacteria of luciferase autoinducer, a molecule involved in the positive transcriptional regulation of the V. fischeri lux operon. Such an absence of visible light production outside of the symbiotic association has not been previously reported among light organ symbionts of this or any other species of luminous bacteria. Levels of luminescence approaching those of the E. scolopes bacteria in the intact association can be restored by the addition of exogenous autoinducer to bacteria in laboratory culture and are affected by the presence of cyclic AMP. We conclude that some condition(s) specific to the internal environment of the light organ is necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.
Mutations leading to constitutive expression from the TOL plasmid meta-cleavage pathway operon are located at the C-terminal end of the positive regulator protein XylS
Authors: Zhou L M , Timmis K N , Ramos J L
Published: November 2016

The XylS protein is the positive activator of the TOL plasmid meta-cleavage pathway operon for the metabolism of alkylbenzoates in Pseudomonas putida. The regulator stimulates transcription from the TOL meta pathway operon promoter (Pm) when activated by benzoate effectors or in the absence of effectors when overproduced. xylS mutant alleles that encode regulators which constitutively mediate expression from Pm were isolated and characterized. The mutant proteins all exhibit single amino acid substitutions adjacent to putative alpha-helix-turn-alpha-helix domains at their C-terminal ends. The XylS mutant proteins can still be partially activated by the usual and unusual benzoate effectors for the wild-type regulator and when activated stimulate higher levels of transcription from Pm.
The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin
Authors: Pepe J C , Miller V L
Published: November 2016

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.
Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis
Authors: Richardson M A , Kuhstoss S , Huber M L , Ford L , Godfrey O , Turner J R , Rao R N
Published: November 2016

Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.
Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli
Authors: Bell P E , Nau C D , Brown J T , Konisky J , Kadner R J
Published: November 2016

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 10----glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.
DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum
Authors: Huang J H , Schell M A
Published: November 2016

The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.
Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph
Authors: Altboum Z , Gottlieb S , Lebens G A , Polacheck I , Segal E
Published: November 2016

Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.
Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production
Authors: Xu P L , Iwata M , Leong S , Sequeira L
Published: November 2016

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.
The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus
Authors: Kovacevic S , Tobin M B , Miller J R
Published: November 2016

Isopenicillin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined. By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the beta-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment. The epimerase gene (cefD) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously. cefD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity. Western immunoblots demonstrated that a protein comigrated with purified S. clavuligerus epimerase at 44 kilodaltons. cefD and cefE were separated by an 81-base-pair segment. The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region. Primer extension analysis of S. clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease S1 protection assays showed that a single message may code for epimerase, expandase, and another unknown protein. When cefD and cefE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E. coli.
Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis
Authors: Slock J , VanRiet D , Kolibachuk D , Greenberg E P
Published: November 2016

Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing a tac promoter-controlled luxR was mutagenized in vitro with hydroxylamine, and luxR mutant plasmids were identified by their inability to complement a luxR deletion mutation in trans. Sixteen luxR mutant plasmids were obtained, ten of which encoded full-length but inactive luxR gene products as demonstrated by a Western immunoblot analysis. The effects of 1 of the 10 mutations could be overcome by the addition of autoinducer at a high concentration. The mutations in each of the 10 mutant plasmids that directed the synthesis of an inactive LuxR protein were identified by DNA sequencing. Of the 10 proteins encoded by the mutant luxR plasmids, 9 differed from the normally active LuxR in only a single amino acid residue. The amino acid residue substitutions in the proteins encoded by the nine mutant luxR genes clustered in two regions. One region around the middle of the polypeptide encoded by luxR was hypothesized to represent an autoinducer-binding domain, and the other region towards the carboxy terminus of the gene product was hypothesized to constitute a lux operator DNA-binding domain or a lux operator DNA recognition domain.
Prey-derived signals regulating duration of the developmental growth phase of Bdellovibrio bacteriovorus
Authors: Gray K M , Ruby E G
Published: November 2016

The filamentous elongation typical of growth-phase cells of the predatory bacterium Bdellovibrio bacteriovorus is mediated by regulatory signals that are derived from the prey cell itself. These signals regulate the differentiation of growth-phase cells into the attack phase and appear to be required for continued filamentous growth by prey-dependent wild-type bdellovibrios and their prey-independent mutant derivatives alike. Using a prey-independent bdellovibrio strain, we have developed an assay for the detection and quantification of the growth-extending signal activity present in extracts of prey cells. This prey-derived regulatory activity was shown to be independent of its nutritional contribution to the bdellovibrios and was found to occur in heat-stable, proteinlike compounds of a variety of native molecular weights within the soluble fraction of extracts from both gram-negative and gram-positive bacteria.
Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation
Authors: Watanabe T , Oyanagi W , Suzuki K , Tanaka H
Published: November 2016

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.
Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362
Authors: Broadwell A H , Clark M A , Baumann L , Baumann P
Published: November 2016

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.
Suppression of early competence mutations in Bacillus subtilis by mec mutations
Authors: Roggiani M , Hahn J , Dubnau D
Published: November 2016

Although competence normally develops only in glucose-minimal salts media, mecA and mecB mutations permit the expression of competence and of late competence genes in complex media as well (D. Dubnau and M. Roggiani, J. Bacteriol. 172:4048-4055, 1990). The expression of late competence genes is dependent on the products of the regulatory genes comA, comB, comP, sin, abrB, spo0H, and spo0A. We show here that this list must be extended to include degU, csh-293, and spo0K. mecA and -B mutations bypass most of these requirements, making the expression of late competence genes and of competence itself independent of all of these regulatory genes, with the exceptions of spo0A and spo0K (in the case of mecB). The expression of late competence genes in mec mutants that are deficient for each of the bypassed regulatory functions is still under growth stage-specific regulation. The implications of these findings are discussed, and a provisional scheme for the flow of information during the development of competence is proposed.
Characterization of the light-regulated operon encoding the phycoerythrin-associated linker proteins from the cyanobacterium Fremyella diplosiphon
Authors: Federspiel N A , Grossman A R
Published: November 2016

Many biological processes in photosynthetic organisms can be regulated by light quantity or light quality or both. A unique example of the effect of specific wavelengths of light on the composition of the photosynthetic apparatus occurs in cyanobacteria that undergo complementary chromatic adaptation. These organisms alter the composition of their light-harvesting organelle, the phycobilisome, and exhibit distinct morphological features as a function of the wavelength of incident light. Fremyella diplosiphon, a filamentous cyanobacterium, responds to green light by activating transcription of the cpeBA operon, which encodes the pigmented light-harvesting component phycoerythrin. We have isolated and determined the complete nucleotide sequence of another operon, cpeCD, that encodes the linker proteins associated with phycoerythrin hexamers in the phycobilisome. The cpeCD operon is activated in green light and expressed as two major transcripts with the same 5' start site but differing 3' ends. Analysis of the kinetics of transcript accumulation in cultures of F. diplosiphon shifted from red light to green light and vice versa shows that the cpeBA and cpeCD operons are regulated coordinately. A common 17-base-pair sequence is found upstream of the transcription start sites of both operons. A comparison of the predicted amino acid sequences of the phycoerythrin-associated linker proteins CpeC and CpeD with sequences of other previously characterized rod linker proteins shows 49 invariant residues, most of which are in the amino-terminal half of the proteins.
Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences
Authors: Prère M F , Chandler M , Fayet O
Published: November 2016

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.
Transcriptional induction of purple membrane and gas vesicle synthesis in the archaebacterium Halobacterium halobium is blocked by a DNA gyrase inhibitor
Authors: Yang C F , DasSarma S
Published: November 2016

We have investigated the expression of the bacteriorhodopsin gene (bop) and the gas vesicle protein gene (gvpA) in the extremely halophilic archaebacterium Halobacterium halobium, using primer-directed reverse transcription of RNA to quantify message levels. The level of gvpA gene transcript was found to increase about 5-fold from early to mid-logarithmic growth phase, while the level of bop gene transcript increased about 20-fold from mid-logarithmic to stationary phase. Transcriptional induction of both the gvpA and bop genes was significantly reduced by aeration and almost completely blocked by the DNA gyrase inhibitor novobiocin.
Adenine deaminase and adenine utilization in Saccharomyces cerevisiae
Authors: Deeley M C
Published: November 2016

Compared with other purine salvage and nitrogen catabolism enzymatic activities, adenine deaminase (adenine aminohydrolase [AAH]; EC 3.5.4.2) activity in Saccharomyces cerevisiae is uniquely regulated. AAH specific activity is not induced by adenine and is reduced sevenfold when cells are cultivated in medium containing proline in place of ammonium as the sole nitrogen source. Exogenous adenine enters metabolic pathways primarily via the function of either AAH or adenine phosphoribosyltransferase (APRT; EC 2.4.2.7). Exogenous adenosine cannot normally be utilized as a purine source. Strains efficiently utilized adenosine or inosine when grown in pH 4.5 medium containing Triton X-100. A recessive mutation permitting utilization of adenosine or inosine in standard media was isolated. In both situations, growth of purine auxotrophs required either AAH or APRT activity. With medium containing either ammonium or proline as a nitrogen source, minimum doubling times of purine auxotrophs deficient in either APRT or AAH were measured. In proline-based medium, AAH and APRT permitted equal utilization of exogenous adenine. In ammonium-based medium, the absence of APRT increased the minimum doubling time by 50%. Similar experiments using sufficient exogenous histidine to feedback inhibit histidine biosynthesis failed to affect the growth rates of adenine auxotrophs blocked in AAH or APRT, indicating that the histidine-biosynthetic pathway does not play a significant role in adenine utilization. The gene that encodes AAH in S. cerevisiae was isolated by complementation using yeast strain XD1-1, which is deficient in AAH, APRT, and purine synthesis. A 1.36-kb EcoRI-SphI fragment was demonstrated to contain the structural gene for AAH by expressing this DNA in Escherichia coli under control of the trp promoter-operator. Northern (RNA) studies using the AAH-, APRT-, and CDC3-coding regions indicated that AAH regulation was not mediated at the level of transcription or mRNA degradation.
The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis
Authors: Molenaar D , Bolhuis H , Abee T , Poolman B , Konings W N
Published: November 2016

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.
A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis
Authors: Green C J , Vold B S
Published: November 2016

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.
Conjugative transfer of Enterococcus faecalis plasmid pAD1: nucleotide sequence and transcriptional fusion analysis of a region involved in positive regulation
Authors: Pontius L T , Clewell D B
Published: November 2016

The Enterococcus faecalis plasmid pAD1 undergoes conjugative transfer in response to cAD1, a peptide sex pheromone emitted by potential bacterial recipients. Regulation of pAD1 transfer involves a number of plasmid-encoded determinants:iad, which determines a peptide-competitive inhibitor iAD1; signal sensing and transducing elements; and negative and positive regulators. The key positive regulator(s) of the pheromone response is believed to be encoded within a segment designated the E region of the plasmid. In this study, we analyzed the nucleotide sequence and transcription within the E region. An open reading frame designated traE1 was identified; its inferred protein consists of 118 amino acids. Insertional mutagenesis of traE1 resulted in a complete loss in plasmid transfer capability. Analysis of Tn917-lac insertions giving rise to transcriptional lacZ fusions showed that traE1 is transcribed only under cAD1 inducing conditions. Analysis of additional lacZ fusions within the region provided some insight into the roles of potential regulatory signals within and around the nucleotide sequences reported here. A regulatory role appearing to involve read-through of certain key transcription termination sequences seemed evident.
Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence
Authors: Raymond-Denise A , Guillen N
Published: November 2016

The Bacillus subtilis dinR gene product is homologous to the LexA protein of Escherichia coli and regulates the expression of dinR and dinC. Using transcriptional fusions in the dinR and the recA genes, we have investigated the epistatic relationship between these two genes during the SOS response induced either by DNA damage or by competence. The results show that after DNA damage, induction of the expression of both recA and dinR is dependent on the activity of the DinR and RecA proteins. A RecA-dependent activity on DinR is proposed as the initial event in the induction of the SOS network. In contrast, the competence-related induction of dinR and recA appears to involve two distinct mechanisms. While one mechanism corresponds to the classical regulation of the SOS response, the other appears to involve an activating factor. Moreover, this factor is active in cells in which competence is prevented by a mutation in the regulatory gene comA.
Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis
Authors: Ireton K , Grossman A D
Published: November 2016

The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.
Transcriptional regulation of the ilv-leu operon of Bacillus subtilis
Authors: Grandoni J A , Zahler S A , Calvo J M
Published: November 2016

We used primer extension and mutational analysis to identify a promoter upstream of ilvB, the first gene in the ilv-leu operon of Bacillus subtilis. Between the promoter and ilvB, there is a 482-bp leader region which contains a sequence that resembles a factor-independent transcription terminator. In in vitro transcription experiments, 90% of transcripts initiated at the ilvB promoter ended at a site near this terminator. Primer extension analysis of RNA synthesized in vivo showed that the steady-state level of mRNA upstream of the terminator was twofold higher from cells limited for leucine than it was from cells grown with excess leucine. mRNA downstream of the terminator was 14-fold higher in cells limited for leucine than in cells grown with excess leucine. Measurement of mRNA degradation rates showed that the half-life of ilv-leu mRNA was the same when the cells were grown with or without leucine. These data demonstrate that the ilv-leu operon is regulated by transcription attenuation.
Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum
Authors: Narberhaus F , Bahl H
Published: November 2016

The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism.