bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribonucleotide Reduction in
Pseudomonas
Species: Simultaneous Presence of Active Enzymes from Different Classes
Authors: Jordan Albert , Torrents Eduard , Sala Irma , Hellman Ulf , Gibert Isidre , Reichard Peter
Published: December 2019

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of
Pseudomonas aeruginosa
contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of
Pseudomonas
also contain the genes for three classes. Extracts from
P. aeruginosa
and
P. stutzeri
grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the
Pseudomonas
class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding
Escherichia coli
enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of
P. aeruginosa
was more related to that of eukaryotes than to that of
E. coli
or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in
Pseudomonas
species.
Type 2 NADH Dehydrogenases in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration
Authors: Howitt Crispin A. , Udall Pacer K. , Vermaas Wim F. J.
Published: December 2019

Analysis of the genome of
Synechocystis
sp. strain PCC 6803 reveals three open reading frames (
slr0851
,
slr1743
, and
sll1484
) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in
Synechocystis
sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames,
slr1743
, was able to functionally complement an
Escherichia coli
mutant lacking both NDH-1s and NDH-2s. Therefore,
slr0851
,
slr1743
, and
sll1484
have been designated
ndbA
,
ndbB
, and
ndbC
, respectively. Strains that lacked one or more of the
ndb
genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of
ndb
genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in
Synechocystis
sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.
Temperature-Sensitive Motility of
Sulfolobus acidocaldarius
Influences Population Distribution in Extreme Environments
Authors: Lewus Paul , Ford Roseanne M.
Published: December 2019

A three-dimensional tracking microscope was used to quantify the effects of temperature (50 to 80°C) and pH (2 to 4) on the motility of
Sulfolobus acidocaldarius
, a thermoacidophilic archaeon. Swimming speed and run time increased with temperature but remained relatively unchanged with increasing pH. These results were consistent with reported changes in the rate of respiration of
S. acidocaldarius
as a function of temperature and pH. Cells exhibited a forward-biased turn angle distribution with a mean of 54°. Cell trajectories during a run were in the shape of right-handed helices. A cellular dynamics simulation was used to test the hypothesis that a population of
S. acidocaldarius
cells could distribute preferentially in a spatial temperature gradient due to variation in swimming speed. Simulation results showed that a population of cells could migrate from a higher to a lower temperature in the presence of sharp temperature gradients. This simulation result was achieved without incorporating the ability of cells to sense a temporal thermal gradient; thus, the response was not thermotactic. We postulate that this temperature-sensitive motility is one survival mechanism of
S. acidocaldarius
that allows this organism to move away from lethal hot spots in its hydrothermal environment.
Site-Specific Recombination of Temperate
Myxococcus xanthus
Phage Mx8: Genetic Elements Required for Integration
Authors: Magrini Vincent , Creighton Chad , Youderian Philip
Published: December 2019

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host,
Myxococcus xanthus
, by a mechanism of site-specific recombination. The Mx8
int-attP
genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3′ ends of either of two tandem tRNA
Asp
genes,
trnD1
and
trnD2
, located within the
attB
locus of the
M. xanthus
genome. Although Int-mediated site-specific recombination occurs between
attP
and either
attB1
(within
trnD1
) or
attB2
(within
trnD2
), the
attP × attB1
reaction is highly favored and often is accompanied by a deletion between
attB1
and
attB2
. The
int
gene is the only Mx8 gene required in
trans
for
attP × attB
recombination. The
int
promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate
attP × attB
recombination efficiently. The
attP
core lies within the
int
coding sequence, and the product of integration is a prophage in which the 3′ end of
int
is replaced by host sequences. The prophage
intX
gene is predicted to encode an integrase with a different C terminus.
Site-Specific Recombination of Temperate
Myxococcus xanthus
Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification
Authors: Magrini Vincent , Storms Michael L. , Youderian Philip
Published: December 2019

Temperate
Myxococcus xanthus
phage Mx8 integrates into the
attB
locus of the
M. xanthus
genome. The phage attachment site,
attP
, is required in
cis
for integration and lies within the
int
(integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end of
int
to generate the modified
intX
gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes
attP × attB
recombination more efficiently than
attR × attB
,
attL × attB
, or
attB × attB
recombination. The
attP
and
attB
sites share a common core. Sequences flanking both sides of the
attP
core within the
int
gene are necessary for
attP
function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the
attP
core. Expression of the
uoi
gene immediately upstream of
int
inhibits integrative (
attP × attB
) recombination, supporting the idea that
uoi
encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.
Nitric Oxide Is a Signal for NNR-Mediated Transcription Activation in
Paracoccus denitrificans
Authors: Van Spanning Rob J. M. , Houben Edith , Reijnders Willem N. M. , Spiro Stephen , Westerhoff Hans V. , Saunders Neil
Published: December 2019

By using the ′
lacZ
gene, the activities of the
nirI
,
nirS
, and
norC
promoters were assayed in the wild type and in NNR-deficient mutants of
Paracoccus denitrificans
grown under various growth conditions. In addition, induction profiles of the three promoters in response to the presence of various nitrogenous oxides were determined. Transcription from the three promoters required the absence of oxygen and the presence both of the transcriptional activator NNR and of nitric oxide. The activity of the
nnr
promoter itself was halved after the cells had been switched from aerobic respiration to denitrification. This response was apparently not a result of autoregulation or of regulation by FnrP, since the
nnr
promoter was as active in the wild-type strain as it was in NNR- or FnrP-deficient mutants.
Swarming by
Pseudomonas syringae
B728a Requires
gacS
(
lemA
) and
gacA
but Not the Acyl-Homoserine Lactone Biosynthetic Gene
ahlI
Authors: Kinscherf Thomas G. , Willis David K.
Published: December 2019

Pseudomonas syringae
pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the
gacS
(formerly
lemA
) or
gacA
gene of B728a eliminate the ability of this
P. syringae
isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on
gacS
for swarming. Since
gacS
and
gacA
mutants are known to be deficient in
N
-acyl-
l
-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the
ahlI
gene of B728a. This mutant did not make any acyl-HSL detectable by the
Agrobacterium traG
::
lacZ
reporter system, yet was unaffected in its ability to swarm. Other phenotypes of
gacS
and
gacA
mutations were similarly unaffected in the
ahlI
mutant.
A Novel
Sinorhizobium meliloti
Operon Encodes an α-Glucosidase and a Periplasmic-Binding-Protein-Dependent Transport System for α-Glucosides
Authors: Willis Laura B. , Walker Graham C.
Published: December 2019

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a
Sinorhizobium meliloti
genomic library and discovered a segment of
S. meliloti
DNA which allows
Ralstonia eutropha
to grow on the α-glucosides sucrose, maltose, and trehalose. Tn
5
mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named
agl
, for α-glucoside utilization. Four of these (
aglE
,
aglF
,
aglG
, and
aglK
) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (
aglA
) appears to encode an α-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne
agl
genes permit uptake of radiolabeled sucrose into
R. eutropha
cells. Analysis of the properties of
agl
mutants suggests that
S. meliloti
possesses at least one additional α-glucosidase as well as a lower-affinity transport system for α-glucosides. It is possible that the Fix
+
phenotype of
agl
mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to
aglEFGAK
include a probable regulatory gene (
aglR
),
zwf
and
edd
, which encode the first two enzymes of the Entner-Doudoroff pathway,
pgl
, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel
Rhizobium
-specific repeat element.
The Genes
rubA
and
rubB
for Alkane Degradation in
Acinetobacter
sp. Strain ADP1 Are in an Operon with
estB
, Encoding an Esterase, and
oxyR
Authors: Geißdörfer Walter , Kok Ruben G. , Ratajczak Andreas , Hellingwerf Klaas J. , Hillen Wolfgang
Published: December 2019

Alkanes are oxidized in
Acinetobacter
sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase.
rubA
and
rubB
encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in
rubA
or
rubB
lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes,
estB
and
oxyR
, are located downstream of
rubB
, but are not necessary for alkane degradation.
estB
encodes a functional esterase.
oxyR
encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide.
rubA
,
rubB
,
estB
, and
oxyR
constitute an operon, which is constitutively transcribed from a ς
70
promoter, and an
estB-oxyR
containing message is also transcribed from an internal promoter.
Interaction of
Bacillus subtilis
Fur (Ferric Uptake Repressor) with the
dhb
Operator In Vitro and In Vivo
Authors: Bsat Nada , Helmann John D.
Published: December 2019

Bacillus subtilis
contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (
dhb
) operon. The purified protein binds with high affinity and selectivity to the
dhb
regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the
dhb
promoter by ς
A
RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the
fur
gene reveals that in vivo repression of the
dhb
operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 μM but not at 5 μM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.
Mycobacterium tuberculosis rrn
Promoters: Differential Usage and Growth Rate-Dependent Control
Authors: Verma Anita , Sampla Avinash K. , Tyagi Jaya Sivaswami
Published: December 2019

Mycobacterium tuberculosis
is a slow-growing pathogen and is characterized by a low content of RNA per unit of DNA. rRNAs represent a major proportion of the total RNA pool, and the entire requirement for rRNA is met by transcription from a single
rrn
operon that is driven by two promoters, P1 and P3. This study attempted to analyze the specific role of the
rrn
promoter in determining the characteristically low levels of RNA in
M. tuberculosis
. For this purpose, the activity of the
M. tuberculosis rrn
promoter as a function of the growth rate was studied by
rrn-lacZ
promoter fusion, hybridization, and primer extension analysis in
M. smegmatis. rrn
promoter signals were faithfully recognized in
M. smegmatis
cultures harboring the
rrn-lacZ
promoter construct. In
M. smegmatis
cultures that displayed doubling times varying between 3.06 and 6.5 h, β-galactosidase activity increased ∼sixfold in proportion to the growth rate (μ). There was a corresponding increase in the amount of
lacZ
-specific mRNA, while the plasmid copy number remained essentially unchanged. For any given μ, the P3 promoter was ∼twofold more efficiently utilized than the P1 promoter. Since both promoters of the
M. tuberculosis rrn
operon are regulatable as a function of growth rate in
M. smegmatis
cultures, it is implied that the inherent structure or sequence of the
rrn
promoter per se is not primarily responsible for the observed lack of modulation of RNA synthesis in
M. tuberculosis.
Mutational Analysis of the
cbb
Operon (CO
2
Assimilation) Promoter of
Ralstonia eutropha
Authors: Jeffke Thomas , Gropp Niels-Holger , Kaiser Claudia , Grzeszik Claudia , Kusian Bernhard , Bowien Botho
Published: December 2019

P
L
promoters direct the transcription of the duplicated
cbb
operons from the facultative chemoautotroph
Ralstonia eutropha
H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO
2
assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. P
L
was altered in its presumed hexameric −35 and/or −10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the ς
70
consensus promoter of
Escherichia coli
. P
L
::
lacZ
transcriptional fusions of various promoter variants were assayed in transconjugant strains of
R. eutropha
as well as in corresponding
cbbR
deletion mutants. Mutations increasing the similarity of the −35 and/or −10 box to the consensus sequence stimulated P
L
activity to various extents, whereas mutations deviating from the consensus decreased the activity. The length of the spacer region also proved to be critical. The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active P
L
. All improved P
L
mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive. They were also activated in the
cbbR
mutants. The activity of the overlapping, divergently oriented
cbbR
promoter was less affected by the mutations. The half- and full-consensus P
L
mutants were comparably active in
E. coli
. Two major conclusions were drawn from the results: (i) the location and function of P
L
were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of the
R. eutropha cbb
operons.
Biochemical and Genetic Evidence for Participation of DevR in a Phosphorelay Signal Transduction Pathway Essential for Heterocyst Maturation in
Nostoc punctiforme
ATCC 29133
Authors: Hagen Kari D. , Meeks John C.
Published: December 2019

In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in
Nostoc punctiforme
ATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate
N. punctiforme
mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox
−
phenotype like the original
devR
insertion mutant UCD 311, consistent with a phosphotransferase role for DevR.
Isolation and Properties of the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL
Authors: Money Tracy , Jones Tamera , Dixon Ray , Austin Sara
Published: December 2019

In
Azotobacter vinelandii
, activation of
nif
gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.
Genomic Relatedness of
Chlamydia
Isolates Determined by Amplified Fragment Length Polymorphism Analysis
Authors: Meijer Adam , Morré Servaas A. , Van Den Brule Adriaan J. C. , Savelkoul Paul H. M. , Ossewaarde Jacobus M.
Published: December 2019

The genomic relatedness of 19
Chlamydia pneumoniae
isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21
Chlamydia trachomatis
isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6
Chlamydia psittaci
isolates (5 avian isolates and 1 feline isolate), and 1
Chlamydia pecorum
isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical
C. trachomatis
isolates. The fingerprints of all
C. pneumoniae
isolates were nearly identical, clustering together at a Dice similarity of 92.6% (± 1.6% standard deviation). The fingerprints of the
C. trachomatis
isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The
C. psittaci
fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of
C. pecorum
was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular,
omp1
and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of
C. pneumoniae
is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.
Cloning, Sequence, and Transcriptional Regulation of the Operon Encoding a Putative N -Acetylmannosamine-6-Phosphate Epimerase (
nanE
) and Sialic Acid Lyase (
nanA
) in
Clostridium perfringens
Authors: Walters Dana M. , Stirewalt Veronica L. , Melville Stephen B.
Published: December 2019

Clostridium perfringens
can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and
N
-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts
N
-acetylmannosamine-6-phosphate to
N
-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from
C. perfringens
NCTC 8798 that contains the
nanE
and
nanA
genes has been cloned. The identification of the
nanA
gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a
nanA Escherichia coli
strain, EV78. The
nanA
gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the
nanE
and
nanA
genes comprise an operon and that transcription of the operon in
C. perfringens
is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of
nanE-nanA
transcription by glucose. With a plasmid construct containing a promoterless
cpe-gusA
gene fusion, in which β-glucuronidase activity indicated that the
gusA
gene acted as a reporter for transcription, a promoter was localized to the region upstream of the
nanE
gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the
nanE
coding sequence.
Functional Analysis of the Carbohydrate-Binding Domains of
Erwinia chrysanthemi
Cel5 (Endoglucanase Z) and an
Escherichia coli
Putative Chitinase
Authors: Simpson Helen D. , Barras Frederic
Published: December 2019

The Cel5 cellulase (formerly known as endoglucanase Z) from
Erwinia chrysanthemi
is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBD
Cel5
has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBD
Cel5
exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from
Escherichia coli
. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the
E. coli
chromosome and purified by metal chelating chromatography. While CBD
Cel5
bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.
Effects of Growth Conditions on Expression of Mycobacterial
murA
and
tyrS
Genes and Contributions of Their Transcripts to Precursor rRNA Synthesis
Authors: Gonzalez-y-Merchand J. A. , Colston M. J. , Cox R. A.
Published: December 2019

All mycobacteria studied to date have an rRNA operon, designated
rrnA
, located downstream from a single copy of the
murA
gene, which encodes an enzyme (EC
2.5.1.7
) important for peptidoglycan synthesis. The
rrnA
operon has a promoter, P1(A), located within the coding region of
murA
, near the 3′ end. Samples of RNA were isolated from
Mycobacterium tuberculosis
at different stages of the growth cycle and from
Mycobacterium smegmatis
grown under different conditions. RNase protection assays were used to investigate transcripts of both
murA
and
rrnA
. Transcription of
murA
was found to continue into the 16S rRNA gene, as if
murA
and
rrnA
form a hybrid (protein coding-rRNA coding) operon. During the growth of
M. tuberculosis
, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of
M. smegmatis
RNA revealed that the level of
murA
RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for
murA
and
rrnA
.
M. smegmatis
has a second rRNA operon,
rrnB
, located downstream from a single copy of the
tyrS
gene, encoding tyrosyl-tRNA synthetase. Transcription of
tyrS
was found to continue into the 16S rRNA gene
rrnB
. The hybrid
tyrS-rrnB
operon contributed 0.2 to 0.6% to
rrnB
transcripts. The pattern of
tyrS
expression during the growth cycle matched the pattern of
rrnB
expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.
Three Distinct Phases of Isoprene Formation during Growth and Sporulation of
Bacillus subtilis
Authors: Wagner William P. , Nemecek-Marshall Michele , Fall Ray
Published: December 2019

During growth on a glucose-tryptone medium,
Bacillus subtilis
6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1,3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.
An Unspliced Group I Intron in 23S rRNA LinksChlamydiales, Chloroplasts, and Mitochondria
Authors: Everett Karin D. E. , Kahane Simona , Bush Robin M. , Friedman Maureen G.
Published: December 2019

Chlamydia was the only genus in the orderChlamydiales until the recent characterization ofSimkania negevensis ZT and Parachlamydia acanthamoebae strains. The present study ofChlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU · 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.
The icfG Gene Cluster ofSynechocystis sp. Strain PCC 6803 Encodes an Rsb/Spo-Like Protein Kinase, Protein Phosphatase, and Two Phosphoproteins
Authors: Shi Liang , Bischoff Kenneth M. , Kennelly Peter J.
Published: December 2019

A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered aroundicfG, a gene implicated in the regulation of carbon metabolism, in the genome ofSynechocystissp. strain PCC 6803.slr1860is the ORF foricfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX fromBacillus subtilis. Bracketingslr1860/icfGare (i) ORFslr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases fromB. subtilis, and (ii) ORFsslr1856andslr1859, whose predicted products resemble the respective phosphoprotein substrates for theB. subtilisprotein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed inEscherichia colias a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORFslr1861encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.
An Iron-Regulated Alkyl Hydroperoxide Reductase (AhpC) Confers Aerotolerance and Oxidative Stress Resistance to the Microaerophilic Pathogen Campylobacter jejuni
Authors: Baillon Marie-Louise A. , van Vliet Arnoud H. M. , Ketley Julian M. , Constantinidou Chrystala , Penn Charles W.
Published: December 2019

Microaerophiles likeCampylobacter jejunimust resist oxidative stresses during transmission or infection. Growth ofC. jejuni81116 under iron limitation greatly increased the expression of two polypeptides of 26 and 55 kDa. The identification of these proteins by N-terminal amino acid sequencing showed both to be involved in the defense against oxidative stress. The 55-kDa polypeptide was identical toC. jejunicatalase (KatA), whereas the N terminus of the 26-kDa polypeptide was homologous to a 26-kDaHelicobacter pyloriprotein. The gene encoding theC. jejuni26-kDa protein was cloned, and the encoded protein showed significant homology to the small subunit of alkyl hydroperoxide reductase (AhpC). The upstream region ofahpCencoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences containedflhBandmotBhomologs, which are involved in flagellar motility. There was no evidence for an adjacent homolog ofahpF, encoding the large subunit of alkyl hydroperoxide reductase. Reporter gene studies showed that iron regulation ofahpCandkatAis achieved at the transcriptional level. Insertional mutagenesis of theahpCgene resulted in an increased sensitivity to oxidative stresses caused by cumene hydroperoxide and exposure to atmospheric oxygen, while resistance to hydrogen peroxide was not affected. TheC. jejuniAhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.
Antigen-43-Mediated Autoaggregation ofEscherichia coli Is Blocked by Fimbriation
Authors: Hasman Henrik , Chakraborty Trinad , Klemm Per
Published: December 2019

Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli. Ag43 is responsible for the autoaggregation and flocculation of static liquid cultures of many E. colistrains. The expression of Ag43 has been reported to be phase variable and controlled by the product of the oxyR gene. Type 1 fimbriae are thin adhesive thread-like surface organelles responsible for bacterial receptor recognition and tissue colonization. Like that of Ag43, the expression of type 1 fimbriae is phase variable. Interestingly, previous results have suggested that the expression of type 1 fimbriae and the expression of Ag43 are mutually exclusive. In the present report, we show, by use of well-defined mutants, that fimbriation abolishes Ag43-mediated autoaggregation but does not affect Ag43 expression. Autoaggregation is shown to require an intercellular Ag43-Ag43 interaction, and the physical presence of fimbriae on the cells seems to abrogate this interaction. The Ag43 or OxyR status does not appear to influence fimbria expression, and our results suggest that the expression of Ag43 and the expression of fimbriae are independent processes.
The Periplasmic Murein Peptide-Binding Protein MppA Is a Negative Regulator of Multiple Antibiotic Resistance in Escherichia coli
Authors: Li HongShan , Park James T.
Published: December 2019

MppA is a periplasmic binding protein inEscherichia coliessential for uptake of the cell wall murein tripeptidel-alanyl-γ-d-glutamyl-meso-diaminopimelate. We have found serendipitously thatE. coliK-12 strains carrying a null mutation inmppAexhibit increased resistance to a wide spectrum of antibiotics and to cyclohexane. Normal sensitivity of themppAmutant to these agents is restored bymppAexpressed from a plasmid. As is observed in the multiple antibiotic resistance phenotype inE. colicells, themppAnull mutant overproduces the transcriptional activator, MarA, resulting in expression of the membrane-bound AcrAB proteins that function as a drug efflux pump. Reduced production of OmpF similar to that observed in the multiple antibiotic resistance phenotype is also seen in themppAmutant. These and other data reported herein indicate that MppA functions upstream of MarA in a signal transduction pathway to negatively regulate the expression ofmarAand hence of the MarA-driven multiple antibiotic resistance. Overproduction of cytoplasmic GadA and GadB and of several unidentified cytoplasmic membrane proteins as well as reduction in the amount of the outer membrane protein, OmpP, in themppAnull mutant indicate that MppA regulates a number of genes in addition to those already known to be controlled by MarA.
In Thermoanaerobacterium thermosulfurigenes EM1 S-Layer Homology Domains Do Not Attach to Peptidoglycan
Authors: Brechtel Elke , Bahl Hubert
Published: December 2019

Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins). At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium. The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase. This enzyme was isolated fromT. thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli. The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall. In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T. thermosulfurigenes EM1 were included in these studies. The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi. Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall. Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria.
Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the
n
-Alkane-Assimilating Yeast
Yarrowia lipolytica
Authors: Wang Huijie J. , Le Dall Marie-Thérèse , Waché Yves , Laroche Céline , Belin Jean-Marc , Gaillardin Claude , Nicaud Jean-Marc
Published: December 2019

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the
n
-alkane-assimilating yeast
Yarrowia lipolytica
, encoded by the
POX1
through
POX5
genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of
POX1
only, strains did not grow on fatty acids, whereas
POX4
alone elicited partial growth, and the growth of the double
POX2-POX3
-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the
POX
genotype.
Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation
Authors: Anthony Karen G. , Klimke William A. , Manchak Jan , Frost Laura S.
Published: December 2019

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (
trbA
to
traS
) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F
+
cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.
The Dual-Specificity Protein Phosphatase Yvh1p Acts Upstream of the Protein Kinase Mck1p in Promoting Spore Development in
Saccharomyces cerevisiae
Authors: Beeser Alexander E. , Cooper Terrance G.
Published: December 2019

Diploid
Saccharomyces cerevisiae
cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen. We show that
yvh1
disruption causes a defect in spore maturation; overexpression of
MCK1
or
IME1
suppresses this
yvh1
phenotype. While
mck1
mutations are epistatic to those in
yvh1
relative to spore maturation, overexpression of
MCK1
does not suppress the
yvh1
slow-vegetative-growth phenotype. We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth.
Translation of Two Nested Genes in Bacteriophage P4 Controls Immunity-Specific Transcription Termination
Authors: Forti Francesca , Polo Simona , Lane Kirk B. , Six Erich W. , Sironi Gianpiero , Dehò Gianni , Ghisotti Daniela
Published: December 2019

In phage P4, transcription of the left operon may occur from both the constitutive P
LE
promoter and the regulated P
LL
promoter, about 400 nucleotides upstream of P
LE
. A strong Rho-dependent termination site,

t
imm

, is located downstream of both promoters. When P4 immunity is expressed, transcription starting at P
LE
is efficiently terminated at

t
imm

, whereas transcription from P
LL
is immunity insensitive and reads through

t
imm

. We report the identification of two nested genes,
kil
and
eta
, located in the P4 left operon. The P4
kil
gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the P
LE
promoter, and its expression is controlled by P4 immunity. Overexpression of
kil
causes cell killing. This gene is the terminal part of a longer open reading frame,
eta
, which begins upstream of P
LE
. The
eta
gene is expressed when transcription starts from the P
LL
promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both
kil
and
eta
overlap the

t
imm

site. By cloning
kil
upstream of a tRNA reporter gene, we demonstrated that translation of the
kil
region prevents premature transcription termination at

t
imm

. This suggests that P4 immunity might negatively control
kil
translation, thus enabling transcription termination at

t
imm

. Transcription starting from P
LL
proceeds through

t
imm

. Mutations that create nonsense codons in
eta
caused premature termination of transcription starting from P
LL
. Suppression of the nonsense mutation restored transcription readthrough at

t
imm

. Thus, termination of transcription from P
LL
is prevented by translation of
eta
.
Analysis of BvgA Activation of the Pertactin Gene Promoter in
Bordetella pertussis
Authors: Kinnear Susan M. , Boucher Philip E. , Stibitz Scott , Carbonetti Nicholas H.
Published: December 2019

Bordetella pertussis
, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the
bvg
regulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated.
fha
is transcribed at 10 min following an inducing signal, while
ptx
is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that
prn
is transcribed at 1 h, an intermediate time compared to those of
fha
and
ptx
. We have identified
cis
-acting sequences necessary for expression of
prn
in
B. pertussis
by using
prn-lac
fusions containing alterations in the sequence upstream of the
prn
open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of
prn
to activate transcription from the promoter. Our genetic data indicate that the region critical for
prn
activation extends upstream to position −84. However, these data do not support the location of the
prn
transcription start site as previously published. We used a number of methods, including
prn-lac
fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify the
bvg
-dependent 5′ end of the
prn
transcript to the cytosine at −125 with respect to the published start site.
HWP1
Functions in the Morphological Development of
Candida albicans
Downstream of
EFG1
,
TUP1
, and
RBF1
Authors: Sharkey Laura L. , McNemar Mark D. , Saporito-Irwin Susan M. , Sypherd Paul S. , Fonzi William A.
Published: December 2019

The morphological plasticity of
Candida albicans
is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent.
HWP1
, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of
HWP1
resulted in a medium-conditional defect in hyphal development.
HWP1
expression was blocked in a Δ
efg1
mutant, reduced in an Δ
rbf1
mutant, and derepressed in a Δ
tup1
mutant. Therefore,
HWP1
functions downstream of the developmental regulators
EFG1
,
TUP1
, and
RBF1
. Mutation of
CPH1
had no effect on
HWP1
expression, suggesting that the positive regulators of hyphal development,
CPH1
and
EFG1
, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene,
ECE1
, was similarly regulated by
EFG1
. Since
ECE1
is not required for hyphal development, the regulatory role of
EFG1
apparently extends beyond the control of cell shape determinants. However, expression of
ECE1
was not influenced by
TUP1
, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.