bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane Association and Multimerization of Secreton Component PulC
Authors: Possot Odile M. , Gérard-Vincent Manon , Pugsley Anthony P.
Published: December 2019

The PulC component of the
Klebsiella oxytoca
pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.
Specific Chromosome Alterations in Fluconazole-Resistant Mutants of
Candida albicans
Authors: Perepnikhatka Valentina , Fischer Frank J. , Niimi Masakazu , Baker Rachel A. , Cannon Richard D. , Wang Ying-Kai , Sherman Fred , Rustchenko Elena
Published: December 2019

The exposure of
Candida albicans
to fluconazole resulted in the nondisjunction of two specific chromosomes in 17 drug-resistant mutants, each obtained by an independent mutational event. The chromosomal changes occurred at high frequencies and were related to the duration of the drug exposure. The loss of one homologue of chromosome 4 occurred after incubation on a fluconazole medium for 7 days. A second change, the gain of one copy of chromosome 3, was observed after exposure for 35 or 40 days. We found that the mRNA levels of
ERG11
,
CDR1
,
CDR2
, and
MDR1
, the candidate fluconazole resistance genes, remained either the same or were diminished. The lack of overexpression of putative drug pumps or the drug target indicated that some other mechanism(s) may be operating. The fluconazole resistance phenotype, electrophoretic karyotypes, and transcript levels of mutants were stable after growth for 112 generations in the absence of fluconazole. This is the first report to demonstrate that resistance to fluconazole can be dependent on chromosomal nondisjunction. Furthermore, we suggest that a low-level resistance to fluconazole arising during the early stages of clinical treatment may occur by this mechanism. These results support our earlier hypothesis that changes in
C. albicans
chromosome number is a common means to control a resource of potentially beneficial genes that are related to important cellular functions.
Cloning and Expression of
cadD
, a New Cadmium Resistance Gene of
Staphylococcus aureus
Authors: Crupper Scott S. , Worrell Veronica , Stewart George C. , Iandolo John J.
Published: December 2019

A cadmium resistance gene, designated
cadD
, has been identified in and cloned from the
Staphylococcus aureus
plasmid pRW001. The gene is part of a two-component operon which contains the resistance gene
cadD
and an inactive regulatory gene,
cadX*
. A high degree of sequence similarity was observed between
cadD
and the
cadB
-like gene from
S. lugdunensis
, but no significant similarity was found with either
cadA
or
cadB
from the
S. aureus
plasmids pI258 and pII147. The positive regulatory gene
cadX*
is identical to
cadX
from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS
257
. The expression of
cadD
in
S. aureus
and
Bacillus subtilis
resulted in low-level resistance to cadmium; in contrast,
cadA
and
cadB
from
S. aureus
induced higher level resistance. However, when the truncated version of
cadX
contained in pRW001 is complemented in
trans
with
cadX
from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version of
cadX
contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.
ScoC Regulates Peptide Transport and Sporulation Initiation in
Bacillus subtilis
Authors: Koide Akiko , Perego Marta , Hoch James A.
Published: December 2019

Oligopeptides are transported into
Bacillus subtilis
by two ABC transport systems, App and Opp. Transcription of the operon encoding the Opp system was found to occur during exponential growth, whereas the
app
operon was induced at the onset of stationary phase. Transcription of both operons was completely curtailed by overproduction of the ScoC regulator from a multicopy plasmid and was enhanced in strains with the
scoC
locus deleted. ScoC, a member of the MarR family of transcription regulators, is known from previous studies to be a negative regulator of sporulation and of protease production that acts by binding directly to the promoters of the genes it regulates. Since peptide transport is essential for inactivation of the negative regulation of sporulation by Rap phosphatases, the control of ScoC transcription repression activity plays a crucial role in the initiation of sporulation.
Molecular Cloning and Characterization of a Locus Responsible for O Acetylation of the O Polysaccharide of
Legionella pneumophila
Serogroup 1 Lipopolysaccharide
Authors: Zou Chang Hua , Knirel Yuriy A. , Helbig Jürgen H. , Zähringer Ulrich , Mintz Clifford S.
Published: December 2019

Complementation experiments, Tn
5
mutagenesis, and DNA sequencing were used to identify a locus (
lag-1
) that participates in acetylation of
Legionella pneumophila
serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of lipopolysaccharides from mutant and complemented strains suggest that
lag-1
is responsible for O acetylation of serogroup 1 O polysaccharide.
The Genes Encoding Formamidopyrimidine and MutY DNA Glycosylases in
Escherichia coli
Are Transcribed as Part of Complex Operons
Authors: Gifford Christine M. , Wallace Susan S.
Published: December 2019

Escherichia coli
formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine. The genes encoding these proteins,
fpg
and
mutY
, are both cotranscribed as part of complex operons.
fpg
is the terminal gene in an operon with the gene order
radC
,
rpmB
,
rpmG
, and
fpg
. This operon has transcription initiation sites upstream of
radC
, in the
radC
coding region, and immediately upstream of
fpg
. There is a strong attenuator in the
rpmG-fpg
intergenic region and three transcription termination sites downstream of
fpg
. There is an additional site, in the
radC-rpmB
intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site.
mutY
is the first gene in an operon with the gene order
mutY
,
yggX
,
mltC
, and
nupG
. This operon has transcription initiation sites upstream of
mutY
, in the
mutY
coding region, and immediately upstream of
nupG
. There also appear to be attenuators in the
yggX-mltC
and
mltC-nupG
intergenic regions. The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms.
A
Vibrio cholerae
LysR Homolog, AphB, Cooperates with AphA at the
tcpPH
Promoter To Activate Expression of the ToxR Virulence Cascade
Authors: Kovacikova Gabriela , Skorupski Karen
Published: December 2019

We describe here a new member of the LysR family of transcriptional regulators, AphB, which is required for activation of the
Vibrio cholerae
ToxR virulence cascade. AphB activates the transcription of the
tcpPH
operon in response to environmental stimuli, and this process requires cooperation with a second protein, AphA. The expression of neither
aphA
or
aphB
is strongly regulated by environmental stimuli, raising the possibility that the activities of the proteins themselves may be influenced under various conditions. Strains of the El Tor biotype of
V. cholerae
typically exhibit lower expression of ToxR-regulated virulence genes in vitro than classical strains and require specialized culture conditions (AKI medium) to induce high-level expression. We show here that expression of
aphB
from the
tac
promoter in El Tor biotype strains dramatically increases virulence gene expression to levels similar to those observed in classical strains under all growth conditions examined. These results suggest that AphB plays a role in the differential regulation of virulence genes between the two disease-causing biotypes.
Genetic Organization of the
citCDEF
Locus and Identification of
mae
and
clyR
Genes from
Leuconostoc mesenteroides
Authors: Bekal-Si Ali Sadjia , Diviès Charles , Prévost Hervé
Published: December 2019

In this paper, we describe two open reading frames coding for a NAD-dependent malic enzyme (
mae
) and a putative regulatory protein (
clyR
) found in the upstream region of
citCDEFG
of
Leuconostoc mesenteroides
subsp.
cremoris
195. The transcriptional analysis of the citrate lyase locus revealed one polycistronic mRNA covering the
mae
and
citCDEF
genes. This transcript was detected only on RNA prepared from cells grown in the presence of citrate. Primer extension experiments suggest that
clyR
and the citrate lyase operon are expressed from a bidirectional A-T-rich promoter region located between
mae
and
clyR.
Physicochemical Evidence that
Treponema pallidum
TroA Is a Zinc-Containing Metalloprotein That Lacks Porin-Like Structure
Authors: Deka Ranjit K. , Lee Yong-Hwan , Hagman Kayla E. , Shevchenko Dmitriy , Lingwood Clifford A. , Hasemann Charles A. , Norgard Michael V. , Radolf Justin D.
Published: December 2019

Although TroA (Tromp1) was initially reported to be a
Treponema pallidum
outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein’s amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive β-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.
Methanobacterium thermoautotrophicum
RNA Polymerase and Transcription In Vitro
Authors: Darcy Trevor J. , Hausner Winfried , Awery Donald E. , Edwards Aled M. , Thomm Michael , Reeve John N.
Published: December 2019

RNA polymerase (RNAP) purified from
Methanobacterium thermoautotrophicum
ΔH has been shown to initiate transcription accurately in vitro from the
hmtB
archaeal histone promoter with either native or recombinant forms of the
M. thermoautotrophicum
TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the
M. thermoautotrophicum
RNAP and TFB has been demonstrated.
Isolation of Additional Bacteriophages with Genomes of Segmented Double-Stranded RNA
Authors: Mindich Leonard , Qiao Xueying , Qiao Jian , Onodera Shiroh , Romantschuk Martin , Hoogstraten Deborah
Published: December 2019

Eight different bacteriophages were isolated from leaves of
Pisum sativum
,
Phaseolus vulgaris
,
Lycopersicon esculentum
,
Daucus carota sativum
,
Raphanus sativum
, and
Ocimum basilicum
. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage φ6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of φ6. Three of the viruses have little or no base sequence identity to φ6. Two of the viruses, φ8 and φ12, contain proteins with a size distribution very different from that of φ6 and do not package genomic segments of φ6. Whereas φ6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in
Escherichia coli
or
Salmonella typhimurium
. One of the isolates, φ8, can form plaques on heptoseless strains of
S. typhimurium
.
Citromicrobium bathyomarinum
, a Novel Aerobic Bacterium Isolated from Deep-Sea Hydrothermal Vent Plume Waters That Contains Photosynthetic Pigment-Protein Complexes
Authors: Yurkov Vladimir V. , Krieger Steven , Stackebrandt Erko , Beatty J. Thomas
Published: December 2019

We have taxonomically and phylogenetically characterized a new aerobic bacterial strain (JF-1) that contains photosynthetic pigment-protein complexes and which was recently isolated from black smoker plume waters of the Juan de Fuca Ridge. Strain JF-1 is a gram-negative, yellow-pigmented, motile bacterium that is salt-, pH-, and thermotolerant. These properties are consistent with an oligotrophic adaptation to varied environmental conditions thought to exist around deep-sea hydrothermal vents. The analysis of 16S rDNA sequences revealed that strain JF-1 forms a separate phylogenetic branch between the genus
Erythromonas
and the
Erythromicrobium-Porphyrobacter-Erythrobacter
cluster within the α subclass of the
Proteobacteria
. The taxonomic name
Citromicrobium bathyomarinum
(gen. nov., sp. nov.) is proposed for strain JF-1.
Molecular Cloning, Sequence Analysis, and Characterization of a Penicillin-Resistant
dd
-Carboxypeptidase of
Myxococcus xanthus
Authors: Kimura Yoshio , Takashima Yukie , Tokumasu Yushi , Sato Masayuki
Published: December 2019

We have cloned a gene,
pdcA
, from the genomic library of
Myxococcus xanthus
with an oligonucleotide probe representing conserved regions of penicillin-resistant
dd
-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted
pdcA
gene product showed significant sequence similarity to
N
-acetylmuramoyl-
l
-alanine amidase and penicillin-resistant
dd
-carboxypeptidase, respectively. The
pdcA
gene was expressed in
Escherichia coli
, and the characteristics of the gene product were similar to those of
dd
-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in
pdcA
deletion mutants.
Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents
Authors: Moukadiri Ismaïl , Jaafar Lahcen , Zueco Jesús
Published: December 2019

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of aSaccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with β-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, andPIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.
Regulation and Adaptation of Glucose Metabolism of the Parasitic Protist Leishmania donovani at the Enzyme and mRNA Levels
Authors: ter Kuile Benno H.
Published: December 2019

Adaptation of the glucose metabolism of Leishmania donovani promastigotes (insect stage) was investigated by simultaneously measuring metabolic rates, enzyme activities, message levels, and cellular parameters under various conditions. Chemostats were used to adapt cells to different growth rates with growth rate-limiting or excess glucose concentrations. L. donovanicatabolized glucose to CO2, succinate, acetate, and pyruvate in ratios that depended on growth rate and glucose availability. Rates of glucose consumption were a linear function of growth rate and were twice as high in excess glucose-grown cells as in glucose-limited organisms. The major end product was CO2, but organic end products were also formed in ratios that varied strongly with growth conditions. The specific activities of the 14 metabolic enzymes measured varied by factors of 3 to 17. Two groups of enzymes adapted specific activities in parallel, but there was no correlation between the groups. The activities of only one group correlated with specific rates of glucose metabolism. Total RNA content per cellular protein varied by a factor of 6 and showed a linear relationship with the rate of glucose consumption. There was no correlation between steady-state message levels and activities of the corresponding enzymes, suggesting regulation at the posttranscriptional level. A comparison of the adaptation of energy metabolism in L. donovani and other species suggests that the energy metabolism ofL. donovani is inefficient but is well suited to the environmental challenges that it encounters during residence in the sandfly, its insect vector.
The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii
Authors: Hales Laura M. , Shuman Howard A.
Published: December 2019

To investigate regulatory networks inLegionella pneumophila, the gene encoding the homolog of theEscherichia colistress and stationary-phase sigma factor RpoS was identified by complementation of anE. coli rpoSmutation. An open reading frame that is approximately 60% identical to theE. coli rpoSgene was identified. Western blot analysis showed that the level ofL. pneumophilaRpoS increased in stationary phase. An insertion mutation was constructed in therpoSgene on the chromosome ofL. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates thatL. pneumophilaRpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host,Acanthamoeba castellanii. These data suggest thatL. pneumophilapossesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role ofrpoSinL. pneumophilais very different from what has previously been reported forE. coli rpoS.
The Pseudomonas aeruginosa Exotoxin A Regulatory Gene, ptxS: Evidence for Negative Autoregulation
Authors: Swanson Britta L. , Colmer Jane A. , Hamood Abdul N.
Published: December 2019

We have previously described aPseudomonas aeruginosagene,ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene,ptxS, which is transcribed divergently fromptxRand interferes with the enhancement of exotoxin A synthesis byptxR. However, the mechanisms through whichptxRand/orptxSare regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine ifP. aeruginosacontains a potential regulatory protein that binds specifically to theptxRorptxSupstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing theptxR-ptxSintergenic region was incubated with the lysate ofP. aeruginosaPAO1. The strongest binding activity was detected with a smaller fragment that represents theptxSupstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream ofptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of theptxSisogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carryingptxSintact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate ofEscherichia coliin whichptxSwas overexpressed from the T7 promoter. The effect of PtxS onptxSexpression was examined by using aptxS-lacZfusion plasmid. The level of β-galactosidase activity produced by PAO1::ptxScarrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within theptxSupstream region.
A Megaplasmid-Borne Anaerobic Ribonucleotide Reductase in Alcaligenes eutrophus H16
Authors: Siedow Anja , Cramm Rainer , Siddiqui Roman A. , Friedrich Bärbel
Published: December 2019

The conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth ofAlcaligenes eutrophusH16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdDandnrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion ofnrdDandnrdGinA. eutrophusabolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant ofA. eutrophuswith a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate thatnrdDandnrdGare cotranscribed and that the translation efficiency ofnrdDis 40-fold higher than that ofnrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.
Proton-Nuclear Magnetic Resonance Analyses of the Substrate Specificity of a β-Ketolase from Pseudomonas putida, Acetopyruvate Hydrolase
Authors: Pokorny Diana , Brecker Lothar , Pogorevc Mateja , Steiner Walter , Griengl Herfried , Kappe Thomas , Ribbons Douglas W.
Published: December 2019

A revised purification of acetopyruvate hydrolase from orcinol-grownPseudomonas putidaORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of ∼38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by1H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three1H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A1H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized π-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg2+bound to the enzyme was made.1H–2H exchange reactions showed the complete, fast equilibration of2H into the C-3 of acetopyruvate chemically; this accounts for the appearance of2H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional1H-NMR in normal1H2O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.
Conserved Cytoplasmic Loops Are Important for both the Transport and Chemotaxis Functions of PcaK, a Protein fromPseudomonas putida with 12 Membrane-Spanning Regions
Authors: Ditty Jayna L. , Harwood Caroline S.
Published: December 2019

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) byPseudomonas putidais mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H+symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates thatP. putidahas a chemoreceptor that differs from the classical chemoreceptors described forEscherichia coliandSalmonella typhimurium.
Demonstration by Mass Spectrometry that Purified Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide
Authors: Blanco David R. , Whitelegge Julian P. , Miller James N. , Lovett Michael A.
Published: December 2019

Purified native Tromp1 was subjected to mass spectrometric analysis in order to determine conclusively whether this protein possesses a cleaved or uncleaved signal peptide. The molecular masses of Tromp1, threeTreponema pallidumlipoproteins, and a bovine serum albumin (BSA) control were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The molecular masses of all of theT. pallidumlipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33,571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in comparison to recombinant Tromp1 following cyanogen bromide cleavage, which further confirmed the identity of Tromp1 and showed that native Tromp1 was not degraded at the carboxy terminus. These studies confirm that Tromp1 is processed and does not contain an uncleaved signal peptide as previously reported.
Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori
Authors: Krishnamurthy Partha , Parlow Mary H. , Schneider John , Burroughs Stephanie , Wickland Catherine , Vakil Nimish B. , Dunn Bruce E. , Phadnis Suhas H.
Published: December 2019

TheHelicobacter pylorigenome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.
In Vivo Observation of Cell Division of Anaerobic Hyperthermophiles by Using a High-Intensity Dark-Field Microscope
Authors: Horn Christian , Paulmann Bernd , Kerlen Gertraude , Junker Norbert , Huber Harald
Published: December 2019

To study growth and cell division of anaerobic hyperthermophilic archaea in vivo, a cultivation technique using glass capillaries was developed. At temperatures of 90 to 98°C, at least 10 successive cell divisions ofPyrodictium abyssiTAG 11 were documented. Cells divide by binary fission. Visualized under a modified dark-field microscope, the formation of cannulae, which finally connected all cells, was observed. The cannulae elongated at 1.0 to 1.5 μm/min and reached final lengths of between 30 and 150 μm. A “snapping division”-like mode of cell fission was discovered forThermoproteus tenax.
Effect of Ionic Strength on Initial Interactions of
Escherichia coli
with Surfaces, Studied On-Line by a Novel Quartz Crystal Microbalance Technique
Authors: Otto Karen , Elwing Hans , Hermansson Malte
Published: December 2019

A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated
Escherichia coli
mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths. This technique enabled us to measure both frequency shifts (Δ
f
), i.e., the increase in mass on the surface, and dissipation shifts (Δ
D
), i.e., the viscoelastic energy losses on the surface. Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process. We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces. The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface. While Δ
f
and Δ
D
per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested. We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested. The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss. Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface. Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.
Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA
Authors: Chernyaeva Natalya S. , Murgola Emanuel J. , Mankin Alexander S.
Published: December 2019

We identified a short RNA fragment, complementary to the
Escherichia coli
23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.
A- and T-Tract-Mediated Intrinsic Curvature in Native DNA between the Binding Site of the Upstream Activator NtrC and the
nifLA
Promoter of
Klebsiella pneumoniae
Facilitates Transcription
Authors: Cheema Amrita Kaur , Choudhury Nirupam Roy , Das H. K.
Published: December 2019

The
nif
promoters of
Klebsiella pneumoniae
must be activated by proteins bound to upstream sequences which are thought to interact with the ς
54
-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the
nifLA
promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the
nifLA
promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the
Escherichia coli lacZ
gene of the DNA fragment containing the
nifLA
promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in
K. pneumoniae
and
E. coli
was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the
nifLA
promoter of
K. pneumoniae.
Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of
Phanerochaete chrysosporium
Authors: Dufrêne Yves F. , Boonaert Christophe J. P. , Gerin Patrick A. , Asther Marcel , Rouxhet Paul G.
Published: December 2019

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus
Phanerochaete chrysosporium
. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 ± 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 ± 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.
Role of the Sporulation Protein BofA in Regulating Activation of the
Bacillus subtilis
Developmental Transcription Factor ς
K
Authors: Resnekov Orna
Published: December 2019

During sporulation, the
Bacillus subtilis
transcription factor ς
K
is activated by regulated proteolytic processing. I have used a system that facilitates the analysis of the contributions of a modified form of the processing enzyme, SpoIVFB-GFP, and the regulatory proteins BofA and SpoIVFA to the conversion of pro-ς
K
to ς
K
. The results show that in the presence of BofA, SpoIVFA levels increase by greater than 20-fold, SpoIVFA is substantially stabilized, and pro-ς
K
processing is inhibited. In addition, enhanced accumulation of the SpoIVFA protein in the absence of BofA (achieved through the use of an
ftsH
null mutation) substantially inhibits pro-ς
K
processing. These results suggest that during growth, increased accumulation of the SpoIVFA protein inhibits the activity of SpoIVFB-GFP and regulates the activation of ς
K
.
Molecular Analysis of the Gene Encoding a Novel Transglycosylative Enzyme from
Alteromonas
sp. Strain O-7 and Its Physiological Role in the Chitinolytic System
Authors: Tsujibo Hiroshi , Kondo Norihiko , Tanaka Keiko , Miyamoto Katsushiro , Baba Nao , Inamori Yoshihiko
Published: December 2019

We purified from the culture supernatant of
Alteromonas
sp. strain O-7 and characterized a transglycosylating enzyme which synthesized β-(1→6)-(GlcNAc)
2
, 2-acetamido-6-
O
-(2-acetamido-2-deoxy-β-
d
-glucopyranosyl)-2-deoxyglucopyranose from β-(1→4)-(GlcNAc)
2
. The gene encoding a novel transglycosylating enzyme was cloned into
Escherichia coli
, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial β-
N
-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported β-
N
-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of β-(1→6)-(GlcNAc)
2
on the induction of chitinase was investigated. β-(1→6)-(GlcNAc)
2
induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)
2
, and (GlcNAc)
3
conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.
Identification of the Ω4499 Regulatory Region Controlling Developmental Expression of a
Myxococcus xanthus
Cytochrome P-450 System
Authors: Fisseha Makda , Biran Dvora , Kroos Lee
Published: December 2019

Ω4499 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
chromosome that fuses
lacZ
expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn
5 lac
Ω4499. The DNA upstream of the Ω4499 insertion has been cloned, and the promoter has been localized. Analysis of the DNA sequence downstream of the promoter revealed one complete open reading frame and a second partial open reading frame that is interrupted by Tn
5 lac
Ω4499. The predicted products of these open reading frames are highly similar to reductase and oxidase components of bacterial cytochrome P-450 systems, which allow catabolism or anabolism of unusual compounds. However, the function of the gene products of the Ω4499 locus remains unclear because
M. xanthus
containing Tn
5 lac
Ω4499 exhibits no apparent defect in growth, developmental aggregation, fruiting body formation, or sporulation. Deletion analysis of the Ω4499 regulatory region showed that multiple DNA elements spanning more than 500 bp upstream of the transcriptional start site contribute to developmental promoter activity. At least two DNA elements, one downstream of −49 bp and one between −49 and −218 bp, boosted activity of the promoter in response to intercellular C signaling. Three sequences in the Ω4499 promoter region, centered at −55, −33, and −1 bp, nearly match a 7-bp sequence found in other C signal-dependent promoters. We propose that these sequences, matching the consensus sequence 5′-CAYYCCY-3′, be called C box sequences, and we speculate that these sequences are
cis
-acting regulatory elements important for the expression of
M. xanthus
genes that depend upon intercellular C signaling during development.
Genomic Plasticity in Natural Populations of
Bordetella pertussis
Authors: Stibitz Scott , Yang Mei-Shin
Published: December 2019

We determined the genomic organization of 14 clinical strains of
Bordetella pertussis
isolated over an 18-month period in Alberta, Canada. The maps of these 14 strains, while demonstrating general similarity of gene order, display a number of examples of genomic rearrangements in the form of large chromosomal inversions.
Isolation, Characterization, and Localization of a Capsule-Associated Gene,
CAP10
, of
Cryptococcus neoformans
Authors: Chang Y. C. , Kwon-Chung K. J.
Published: December 2019

Cryptococcus neoformans
is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene,
CAP10
, which is required for capsule formation. Complementation of the acapsular
cap10
mutant produced an encapsulated strain and the deletion of
CAP10
from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When
CAP10
was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that
CAP10
was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of
CAP10
are modulated by the transcriptional factor
STE12
α. Deletion of
STE12
α downregulated the expression levels of
CAP10
while overexpression of
STE12
α upregulated the expression levels of
CAP10
. Animal model studies indicate that deletion of the
CAP10
gene results in the loss of virulence, and complementation of the acapsular phenotype of
cap10
restores virulence. Thus,
CAP10
is required for capsule formation and virulence.
In Vitro Studies on the Initial Reactions of Anaerobic Ethylbenzene Mineralization
Authors: Johnson Hope A. , Spormann Alfred M.
Published: December 2019

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium
Azoarcus
sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With
p
-benzoquinone as the electron acceptor, cell extracts of
Azoarcus
sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min
−1
[mg of protein]
−1
and an apparent

K
m

for ethylbenzene of approximately 60 μM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (
S
)-(−)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (
S
)-(−)-1-phenylethanol but not of (
R
)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (
S
)-(−)-1-phenylethanol oxidation occurred only in the presence of NAD
+
, suggesting that NAD
+
is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in
Azoarcus
sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.