bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene encoding a novel extracellular metalloprotease in Bacillus subtilis
Authors: Sloma A , Rudolph C F , Rufo G A , Sullivan B J , Theriault K A , Ally D , Pero J
Published: November 2016

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.
Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system
Authors: Crutz A M , Steinmetz M , Aymerich S , Richter R , Le Coq D
Published: November 2016

The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.
Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product
Authors: Miller V L , Bliska J B , Falkow S
Published: November 2016

The ability to enter (invade) eucaryotic cells is a property common to many pathogenic bacteria. Yersinia enterocolitica is a facultative intracellular pathogen whose primary site of multiplication is the reticuloendothelial system. In an effort to understand how Y. enterocolitica crosses the intestinal epithelial cell layer, we previously reported the cloning of two loci from Y. enterocolitica that individually conferred an invasive phenotype to the normally noninvasive Escherichia coli HB101. One of these loci, ail, is encoded by a region of DNA that is less than 650 base pairs. We have identified the ail gene product in maxicells as a 17-kilodalton membrane-associated protein. The Ail protein has been purified, and its N-terminal sequence has been determined. The nucleotide sequence of the ail gene revealed a single unique open reading frame of 178 amino acids. Comparison of amino acid sequences deduced from the gene and obtained by analysis of the purified protein identified the first 23 amino acids as a signal sequence. The site(s) at which transcription initiates on the ail gene was identified by primer extension analysis and shown to be identical in E. coli and Y. enterocolitica. Two small open reading frames downstream of ail were found and shown to exhibit considerable identity to the proposed IS3 transposase.
Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ
Authors: Linn T , St Pierre R
Published: November 2016

An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIII site generates an unchanging 5' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of beta-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (lambda TL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.
F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits
Authors: Riegman N , Kusters R , Van Veggel H , Bergmans H , Van Bergen en Henegouwen P , Hacker J , Van Die I
Published: November 2016

The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified. focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA. Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.
VirA, a coregulator of Ti-specified virulence genes, is phosphorylated in vitro
Authors: Huang Y , Morel P , Powell B , Kado C I
Published: November 2016

High-level expression of a chimeric virA gene was obtained by replacing the first 524 codons of virA with the first half of trpE. The encoded fusion protein was isolated and found to exhibit autokinase activity. Therefore, a kinase domain is in the C-terminal portion of VirA, and protein phosphorylation may be an important feature of VirA function.
Methyl transfer in chemotaxis toward sugars by Bacillus subtilis
Authors: Thoelke M S , Casper J M , Ordal G W
Published: November 2016

Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins. These sugars also caused methanol formation on addition. Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.
Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli
Authors: Bourgaize D B , Phillips T A , VanBogelen R A , Jones P G , Neidhardt F C , Fournier M J
Published: November 2016

During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.
Alkaline phosphatase fusions: sensors of subcellular location
Authors: Manoil C , Mekalanos J J , Beckwith J
Published: November 2016

Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.
Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli
Authors: Szumanski M B , Boyle S M
Published: November 2016

The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH). AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E. coli. Sequencing of a 2.97-kilobase-pair fragment of the E. coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3. ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis. Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4. The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity. The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.
Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism
Authors: Angerer A , Gaisser S , Braun V
Published: November 2016

The cloned sfu region of the Serratia marcescens chromosome confers the ability to grow on iron-limited media to an Escherichia coli K-12 strain that is unable to synthesize a siderophore. This DNA fragment was sequenced and found to contain three genes termed sfuA, sfuB, and sfuC, arranged and transcribed in that order. The sfuA gene encoded a periplasmic polypeptide with calculated molecular weights of 36,154 for the precursor and 33,490 for the mature protein. The sfuB gene product was a very hydrophobic protein with a molecular weight of 56,589. The sfuC gene was found to encode a rather polar but membrane-bound protein with a molecular weight of 36,671 which exhibited strong homology to consensus sequences of nucleotide-binding proteins. The number, structural characteristics, and locations of the SfuABC proteins were typical of a periplasmic-binding-protein-dependent transport mechanism. How Fe3+ is solubilized and taken up across the outer membrane remains an enigma.
pdc1(0) mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1
Authors: Seeboth P G , Bohnsack K , Hollenberg C P
Published: November 2016

The PDC1 gene coding for a pyruvate decarboxylase (PDC; EC 4.1.1.1) was deleted from the Saccharomyces cerevisiae genome. The resulting pdc1(0) mutants were able to grow on glucose and still contained 60 to 70% of the wild-type PDC activity. Two DNA fragments with sequences homologous to that of the PDC1 gene were cloned from the yeast genome. One of the cloned genes (PDC5) was expressed at high rates predominantly in pdc1(0) strains and probably encodes the remaining PDC activity in these strains. Expression from the PDC1 promoter in PDC1 wild-type and pdc1(0) strains was examined by the use of two reporter genes. Deletion of PDC1 led to increased expression of the two reporter genes regardless of whether the fusions were integrated into the genome or present on autonomously replicating plasmids. The results suggested that this effect was due to feedback regulation of the PDC1 promoter-driven expression in S. cerevisiae pdc1(0) strains. The yeast PDC1 gene was expressed in Escherichia coli, leading to an active PDC. This result shows that the PDC1-encoded subunit alone can form an active tetramer without yeast-specific processing steps.
Effects of paraquat on Escherichia coli: differences between B and K-12 strains
Authors: Kitzler J W , Minakami H , Fridovich I
Published: November 2016

Escherichia coli B and K-12 are equally susceptible to the bacteriostatic effects of aerobic paraquat, but they differed strikingly when the lethality of paraquat was evaluated. E. coli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E. coli K-12 did not. This difference depended on the ability of the B strain, but not the K-12 strain, to retain internalized paraquat; the B strain was killed on aerobic tryptic soy-yeast extract plates during the incubation which preceded the counting of colonies. This difference in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by cyanide-resistant respiration after brief exposure to paraquat, washing, and testing in fresh medium. This difference was also shown by using [14C]paraquat. This previously unrecognized difference between E. coli B and K-12 has been the cause of apparently contradictory reports and should lead to some reevaluation of the pertinent literature.
Coregulation of type 12 M protein and streptococcal C5a peptidase genes in group A streptococci: evidence for a virulence regulon controlled by the virR locus
Authors: Simpson W J , LaPenta D , Chen C , Cleary P P
Published: November 2016

Group A streptococci express at least two surface-associated virulence factors, the antiphagocytic M protein and the antichemotactic streptococcal C5a peptidase (SCP). Preliminary evidence suggested that the biosynthesis of these two proteins is coordinately controlled and subject to simultaneous phase variation. To explore this possibility further, a series of phase-switching and phase-locked M- variants were assayed for SCP by enzyme-linked immunosorbent assay inhibition and for SCP-specific mRNA by dot blot hybridization. All M- cultures produced diminished amounts of SCP antigen and specific mRNA, whereas revertants produced quantities equivalent to those of the wild-type M+ culture. A phase-locked strain that harbors a deletion in a region upstream of the M12 and SCP genes, termed the virR locus, failed to produce SCP antigen or SCP-specific transcripts. The SCP-specific transcript produced by M+ bacteria was shown by Northern (RNA) blot hybridization to be 4 kilobases in size, distinguishing it from the transcript which encodes M protein. These data demonstrate that phase switching of both SCP and M12 proteins is at the transcriptional level and that expression is under the control of the upstream virR locus. We propose that the genetic determinants of these proteins and of colony morphology comprise a virulence regulon.
Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168
Authors: Zhang J J , Hu F M , Chen N Y , Paulus H
Published: November 2016

The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.
Negative regulator of sigma G-controlled gene expression in stationary-phase Bacillus subtilis
Authors: Rather P N , Coppolecchia R , DeGrazia H , Moran C P
Published: November 2016

In some media, Bacillus subtilis can maintain a prolonged stationary growth phase; however, in other media, nutrient depletion triggers a complex differentiation that culminates in production of a dormant endospore. This differentiation requires the expression of many genes. We found that during the stationary phase in media in which the cells do not form endospores and do not normally express these sporulation-essential genes, a recessive mutation in spoIIAB caused increased transcription of a set of genes essential for sporulation. Evidently, the wild-type product of spoIIAB acts during the stationary phase to prevent expression of additional sporulation-specific genes.
Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen
Authors: Lantz M S , Allen R D , Bounelis P , Switalski L M , Hook M
Published: November 2016

Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.
Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis
Authors: Hulett F M , Bookstein C , Jensen K
Published: November 2016

Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS. The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics. A total of 62% of the first 22 amino-terminal amino acids were identical. Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases. One alkaline phosphatase was a monomer and the other was a dimer. Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B. subtilis.
Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology
Authors: Amann R I , Krumholz L , Stahl D A
Published: November 2016

Fluorescent-dye-conjugated oligonucleotides were used to classify 14 Fibrobacter strains by fluorescence microscopy. On the basis of partial 16S rRNA sequences of six Fibrobacter strains, four hybridization probes were designed to discriminate between the species Fibrobacter succinogenes and Fibrobacter intestinalis and to identify F. succinogenes subsp. succinogenes. After in situ hybridization to whole cells of the six sequenced strains, epifluorescence microscopy confirmed probe specificity. The four probes were then used to make presumptive species and subspecies assignments of eight additional Fibrobacter strains not previously characterized by comparative sequencing. These assignments were confirmed by comparative sequencing of the 16S rRNA target regions from the additional organisms. Single-mismatch discrimination between certain probe and nontarget sequences was demonstrated, and fluorescent intensity was shown to be enhanced by hybridization to multiple probes of the same specificity. The direct detection of F. intestinalis in mouse cecum samples demonstrated the application of this technique to the characterization of complex natural samples.
Adaptive acidification tolerance response of Salmonella typhimurium
Authors: Foster J W , Hall H K
Published: November 2016

Salmonella typhimurium can encounter a wide variety of environments during its life cycle. One component of the environment which will fluctuate widely is pH. In nature, S. typhimurium can experience and survive dramatic acid stresses that occur in diverse ecological niches ranging from pond water to phagolysosomes. However, in vitro the organism is very sensitive to acid. To provide an explanation for how this organism survives acid in natural environments, the adaptive ability of S. typhimurium to become acid tolerant was tested. Logarithmically grown cells (pH 7.6) shifted to mild acid (pH 5.8) for one doubling as an adaptive procedure were 100 to 1,000 times more resistant to subsequent strong acid challenge (pH 3.3) than were unadapted cells shifted directly from pH 7.6 to 3.3. This acidification tolerance response required protein synthesis and appears to be a specific defense mechanism for acid. No cross protection was noted for hydrogen peroxide, SOS, or heat shock. Two-dimensional polyacrylamide gel electrophoretic analysis of acid-regulated polypeptides revealed 18 proteins with altered expression, 6 of which were repressed while 12 were induced by mild acid shifts. An avirulent phoP mutant was 1,000-fold more sensitive to acid than its virulent phoP+ parent, suggesting a correlation between acid tolerance and virulence. The Mg2(+)-dependent proton-translocating ATPase was also found to play an important role in acid tolerance. Mutants (unc) lacking this activity were unable to mount an acid tolerance response and were extremely acid sensitive. In contrast to these acid-sensitive mutants, a constitutively acid-tolerant mutant (atr) was isolated from wild-type LT2 after prolonged acid exposure. This mutant overexpressed several acidification tolerance response polypeptides. The data presented reveal an important acidification defense modulon with broad significance toward survival in biologically hostile environments.
In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide
Authors: Ostrowski J , Kredich N M
Published: November 2016

The cysteine regulon of Salmonella typhimurium is positively regulated by the CysB protein and an inducer, which can be either O-acetyl-L-serine or N-acetyl-L-serine. In vivo experiments confirmed that sulfide and L-cysteine (supplied as L-cystine) interfere with induction by exogenously supplied O-acetyl-L-serine and also showed the same effects when N-acetyl-L-serine was used as an inducer. In a gel shift assay, purified CysB protein bound specifically to a 278-base-pair DNA fragment containing the S. typhimurium cysJIH promoter region. Binding occurred in the absence of inducer but did not stimulate in vitro transcription initiation, indicating that binding alone is insufficient to cause formation of a transcription initiation complex. Addition of N-acetyl-L-serine or O-acetyl-L-serine was required for transcription initiation and also stimulated binding three- to eightfold. Sulfide inhibited both transcription initiation and binding by interfering with the stimulatory effects of inducer in a competitive manner. These findings indicate that sulfide is an anti-inducer and may explain why full expression of the cysteine regulon requires sulfur limitation. L-Cysteine did not affect in vitro transcription initiation or binding of CysB protein to the cysJIH promoter region. The in vivo effects of L-cysteine may be secondary to its degradation to sulfide by the inducible enzyme cysteine desulfhydrase.
Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU
Authors: Msadek T , Kunst F , Henner D , Klier A , Rapoport G , Dedonder R
Published: November 2016

The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.
Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications
Authors: Essar D W , Eberly L , Hadero A , Crawford I P
Published: November 2016

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.
Rhizobium meliloti nodD genes mediate host-specific activation of nodABC
Authors: Honma M A , Asomaning M , Ausubel F M
Published: November 2016

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.
Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family
Authors: Rothmel R K , Aldrich T L , Houghton J E , Coco W M , Ornston L N , Chakrabarty A M
Published: November 2016

Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.
DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionary divergence
Authors: Hartnett C , Neidle E L , Ngai K L , Ornston L N
Published: November 2016

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The DNA sequence reveals that A. calcoaceticus pca genes, encoding enzymes required for protocatechuate metabolism, are arranged in a single transcriptional unit, pcaEFDBCHG, whereas homologous genes are arranged differently in Pseudomonas putida. The pcaG and pcaH genes represent separate reading frames respectively encoding the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.1.3); previously a single designation, pcaA, had been used to represent DNA encoding this enzyme. The alpha and beta protein subunits appear to share common ancestry with each other and with catechol 1,2-dioxygenases from A. calcoaceticus and P. putida. Marked conservation of amino acid sequence is observed in a region containing two histidyl residues and two tyrosyl residues that appear to ligate iron within each oxygenase. In some regions within the aligned oxygenase sequences, DNA sequences appear to be conserved at a level beyond the extent that might have been demanded by selection at the level of protein. In other regions, divergence of DNA sequences appears to have been achieved by substitution of DNA sequence from one genetic segment into another. The results are interpreted to be the consequence of sequence exchange by gene conversion between slipped strands of DNA during evolutionary divergence; mismatch repair between slipped strands may contribute to the maintenance of DNA sequence in divergent genes.
Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene
Authors: Murphy R C , Gasparich G E , Bryant D A , Porter R D
Published: November 2016

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.
Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities
Authors: Larsson C , Morales C , Gustafsson L , Adler L
Published: November 2016

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.
High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli
Authors: Harris C L
Published: November 2016

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.
Heat shock response in mycoplasmas, genome-limited organisms
Authors: Dascher C C , Poddar S K , Maniloff J
Published: November 2016

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.
O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae
Authors: Chang E C , Kosman D J
Published: November 2016

Mutant strains of the yeast Saccharomyces cerevisiae which lack functional Cu,Zn superoxide dismutase (SOD-1) do not grow aerobically unless supplemented with methionine. The molecular basis of this O2-dependent auxotrophy in one of the mutants, Dscd1-1C, has been investigated. Sulfate supported anaerobic but not aerobic mutant growth. On the other hand, cysteine and homocysteine supported aerobic growth while serine, O-acetylserine, and homoserine did not, indicating that the interconversion of cysteine and methionine (and homocysteine) was not impaired. Thiosulfate (S2O3(2-] and sulfide (S2-) also supported aerobic growth; the activities of thiosulfate reductase and sulfhydrylase in the aerobic mutant strain were at wild-type levels. Although the levels of SO4(2-) and adenosine-5'-sulfate (the first intermediate in the SO4(2-) assimilation pathway) were elevated in the aerobically incubated mutant strain, this condition could be attributed to a decrease in protein synthesis caused by the de facto sulfur starvation and not to a block in the pathway. Therefore, the activation of SO4(2-) (to form 3'-phosphoadenosine-5'-phosphosulfate) appeared to be O2 tolerant. Sulfite reductase activity and substrate concentrations [( NADPH] and [SO3(2-)]) were not significantly different in aerobically grown mutant cultures and anaerobic cultures, indicating that SOD-1- mutant strains could reductively assimilate sulfur oxides. However, the mutant strain exhibited an O2-dependent sensitivity to SO3(2-) concentrations of less than 50 microM not exhibited by any SOD-1+ strain or by SOD-1- strains supplemented with a cytosolic O2(-)-scavenging activity. This result suggests that the aerobic reductive assimilation of SO4(2-) at the level of SO3(2-) may generate a cytotoxic compound(s) which persists in SOD-(1-) yeast strains.
Efficiency factors and ATP/ADP ratios in nitrogen-fixing Bacillus polymyxa and Bacillus azotofixans
Authors: Kanamori K , Weiss R L , Roberts J D
Published: November 2016

The efficiency factor, the number of moles of ATP generated per mole of glucose fermented, was determined in anaerobic, non-carbon-limited N2-fixing cultures of Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Clostridium butyricum through identification and quantitation of the fermentation products by 13C nuclear magnetic resonance spectroscopy and measurement of acetate kinase activities. All three Bacillus species had acetate kinase activities and produced acetate and ethanol as the major fermentation products. The maximum amounts of ATP generated per mole of glucose fermented were 2.70, 2.64, and 2.88 mol in B. polymyxa, B. macerans, and B. azotofixans, respectively, compared with 3.25 mol in C. butyricum. Thus, in the N2-fixing Bacillus species, the efficiency factors are lower than that in C. butyricum. Steady-state ATP/ADP concentration ratios were measured in non-carbon-limited N2-fixing cultures of B. polymyxa and B. azotofixans through separation and quantitation of the adenylates in cell extracts by ion-pair reversed-phase high-performance liquid chromatography. The observed ATP/ADP ratios were 4.5 and 3.8, and estimated energy charges were 0.81 to 0.86 and 0.81 to 0.83, respectively, for B. polymyxa and B. azotofixans. The results suggest that under these growth conditions, the rate of ATP regeneration is adequate to meet the energy requirement for N2 fixation in the Bacillus species, in contrast to N2-fixing Clostridium pasteurianum and Klebsiella pneumoniae, for which substantially lower steady-state ATP/ADP ratios and energy charges have been reported. Implications of the results are discussed in relation to possible differences between Bacillus and Clostridium species in energy requirements for N2 fixation and concomitant ammonia assimilation.
Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames
Authors: Menon N K , Robbins J , Peck H D , Chatelus C Y , Choi E S , Przybyla A E
Published: November 2016

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.
Effect of temperature shifts on gliding motility, adhesion, and fatty acid composition of Cytophaga sp. strain U67
Authors: McGrath C F , Moss C W , Burchard R P
Published: November 2016

Gliding motility and flipping of 25 degrees C-adapted Cytophaga sp. strain U67 were inhibited when the bacteria were shifted to a less than or equal to 12 degrees C environment; motility was not blocked by a shift to 13 degrees C. Bacteria adapted to 4 degrees C were motile over the entire 4 to 25 degrees C temperature range tested. U67 adhesion to the substratum appeared to be unaffected by temperature shifts. Bacteria adapted to 4 degrees C had higher proportions of unsaturated and branched-chain fatty acids than did those grown at 25 degrees C. When 25 degrees C-adapted bacteria were subjected to a gradual temperature decline, the time of reappearance of gliding competence at 4 to 5 degrees C was correlated with these changes in fatty acid composition.
Organization of the replication control region of plasmid ColIb-P9
Authors: Hama C , Takizawa T , Moriwaki H , Urasaki Y , Mizobuchi K
Published: November 2016

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.