bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolic Roles of a
Rhodobacter sphaeroides
Member of the ς
32
Family
Authors: Karls Russell K. , Brooks Jacqueline , Rossmeissl Peter , Luedke Janelle , Donohue Timothy J. 
Published: December 2019

ABSTRACT

We report the role of a gene (
rpoH
) from the facultative phototroph
Rhodobacter sphaeroides
that encodes a protein (ς
37
) similar to
Escherichia coli
ς
32
and other members of the heat shock family of eubacterial sigma factors.
R. sphaeroides
ς
37
controls genes that function during environmental stress, since an
R. sphaeroides
ΔRpoH mutant is ∼30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the ΔRpoH mutant lacks several phenotypes characteristic of
E. coli
cells lacking ς
32
. For example, an
R. sphaeroides
ΔRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of
R. sphaeroides
to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the ΔRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The ΔRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42°C, but it exhibited altered induction kinetics of ∼120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (
rpoD
P
HS
,
groESL
1
, etc.) when aerobically grown ΔRpoH cells were placed at 42°C. Under aerobic conditions, it appears that another sigma factor enables the ΔRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an ΔRpoH mutant, reconstituted Eς
37
, or a holoenzyme containing a 38-kDa protein (ς
38
) each transcribed
E. coli
Eς
32
-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the ΔRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37°C.
The Tryptophanase Gene Cluster of
Haemophilus influenzae
Type b: Evidence for Horizontal Gene Transfer
Authors: Martin Kimberly , Morlin Gregory , Smith Arnold , Nordyke Andrea , Eisenstark Abraham , Golomb Miriam 
Published: December 2019

ABSTRACT

Among strains of
Haemophilus influenzae
, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic
H. influenzae
which are homologous to the tryptophanase (
tna
) operon of
Escherichia coli
. The
tna
genes are located on a 3.1-kb fragment between
nlpD
and
mutS
in the
H. influenzae
type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from
nlpD
, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The
tna
cluster is found at the same map location in all indole-positive strains of
H. influenzae
surveyed and is absent from reference type d and e genomes. In contrast to
H. influenzae
, most other
Haemophilus
species lack
tna
genes. Phylogenetic comparisons suggest that the
tna
cluster was acquired by intergeneric lateral transfer, either by
H. influenzae
or a recent ancestor, and that
E. coli
may have acquired its
tnaA
gene from a related source. Genomes of virulent
H. influenzae
resemble those of pathogenic enterics in having an island of laterally transferred DNA next to
mutS.
Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene (
cpe
) of
Clostridium perfringens
Authors: Zhao Yuling , Melville Stephen B. 
Published: December 2019

ABSTRACT

Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of
Clostridium perfringens
enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the
cpe
promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of
cpe
, translational fusions containing the promoter deletions were made with the
gusA
gene of
Escherichia coli
, which codes for β-glucuronidase;
E. coli-C. perfringens
shuttle vectors carrying the fusions were introduced into
C. perfringens
by electroporation. In addition, in vitro transcription assays were performed with the
cpe
promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of
Bacillus subtilis
, the same compartment in which enterotoxin is synthesized in
C. perfringens.
AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes
Authors: Sadowsky Michael J. , Tong Zhaokun , de Souza Mervyn , Wackett Lawrence P. 
Published: December 2019

ABSTRACT

Pseudomonas
sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding
N
-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway,
atzC
, was cloned from a
Pseudomonas
sp. strain ADP cosmid library as a 25-kb
Eco
RI DNA fragment in
Escherichia coli
. The
atzC
gene was further delimited by functional analysis following transposon Tn
5
mutagenesis and subcloned as a 2.0-kb
Eco
RI-
Ava
I fragment. An
E. coli
strain containing this DNA fragment expressed
N
-isopropylammelide isopropylamino hydrolase activity, metabolizing
N
-isopropylammelide stoichiometrically to cyanuric acid and
N
-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of
E. coli
cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.
Succinoglycan Production by
Rhizobium meliloti
Is Regulated through the ExoS-ChvI Two-Component Regulatory System
Authors: Cheng Hai-Ping , Walker Graham C. 
Published: December 2019

ABSTRACT

The
Rhizobium meliloti exoS
gene is involved in regulating the production of succinoglycan, which plays a crucial role in the establishment of the symbiosis between
R. meliloti
Rm1021 and its host plant, alfalfa. The
exoS96
::Tn
5
mutation causes the upregulation of the succinoglycan biosynthetic genes, thereby resulting in the overproduction of succinoglycan. Through cloning and sequencing, we found that the
exoS
gene is a close homolog of the
Agrobacterium tumefaciens chvG
gene, which has been proposed to encode the sensor protein of the ChvG-ChvI two-component regulatory system, a member of the EnvZ-OmpR family. Further analyses revealed the existence of a newly discovered
A. tumefaciens chvI
homolog located just upstream of the
R. meliloti exoS
gene.
R. meliloti
ChvI may serve as the response regulator of ExoS in a two-component regulatory system. By using ExoS-specific antibodies, it was found that the ExoS protein cofractionated with membrane proteins, suggesting that it is located in the cytoplasmic membrane. By using the same antibodies, it was shown that the
exoS96
::Tn
5
allele encodes an N-terminal truncated derivative of ExoS. The cytoplasmic histidine kinase domain of ExoS was expressed in
Escherichia coli
and purified, as was the
R. meliloti
ChvI protein. The ChvI protein autophosphorylated in the presence of acetylphosphate, and the ExoS cytoplasmic domain fragment autophosphorylated at a histidine residue in the presence of ATP. The ChvI protein was phosphorylated in the presence of ATP only when the histidine kinase domain of ExoS was also present. We propose a model for regulation of succinoglycan production by
R. meliloti
through the ExoS-ChvI two-component regulatory system.
All Seven
comG
Open Reading Frames Are Required for DNA Binding during Transformation of Competent
Bacillus subtilis
Authors: Chung Y. S. , Dubnau D. 
Published: December 2019

ABSTRACT

The seven proteins encoded by the
comG
operon of
Bacillus subtilis
exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in
comG
result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed.
Lagging-Strand Replication from the
ssoA
Origin of Plasmid pMV158 in
Streptococcus pneumoniae
: In Vivo and In Vitro Influences of Mutations in Two Conserved
ssoA
Regions
Authors: Kramer M. Gabriela , Khan Saleem A. , Espinosa Manuel 
Published: December 2019

ABSTRACT

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin,
sso
. We have used the pMV158-derivative plasmid pLS1 (containing the
ssoA
type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the
ssoA
(the recombination site B [RS
B
] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in
Streptococcus pneumoniae
. Cells containing plasmids with mutations in the RS
B
accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS
B
region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.
Identification of an Na
+
-Dependent Malonate Transporter of
Malonomonas rubra
and Its Dependence on Two Separate Genes
Authors: Schaffitzel Christiane , Berg Michael , Dimroth Peter , Pos Klaas M. 
Published: December 2019

ABSTRACT

Two membrane proteins encoded by the malonate fermentation gene cluster of
Malonomonas rubra
, MadL and MadM, have been synthesized in
Escherichia coli
. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na
+
dependent, and imposition of a ΔpNa
+
markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into
E. coli
BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate
−
is the transported malonate species. The stimulation of malonate uptake by Na
+
ions showed Michaelis-Menten kinetics, and a

K
m

for Na
+
of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na
+
/Hmalonate
−
symporter and that it is dependent on two separate genes.
Interactions between the Promoter Regions of Nitrogenase Structural Genes (
nifHDK2
) and DNA-Binding Proteins from N
2
- and Ammonium-Grown Cells of the Archaeon
Methanosarcina barkeri
227
Authors: Chien Yueh-tyng , Helmann John D. , Zinder Stephen H. 
Published: December 2019

ABSTRACT

Transcription initiation in
Archaea
(archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of
nifHDK2
genes, encoding
Methanosarcina barkeri
nitrogenase, are present in N
2
-grown cells but not in ammonium-grown cells, indicating that
nif
transcription is regulated by the nitrogen source. In this study, we detected proteins in
M. barkeri
cell extracts that bind specifically to DNA containing the putative promoter region of
nifHDK2
. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (
mcr
) promoter region DNA and the
nifH2
promoter region DNA competed for a common factor(s). There was no binding to the
nifH2
promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for
mcr
genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the
nif
promoter region by extracts of N
2
-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N
2
-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of
nif
gene expression in
M. barkeri
in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.
Cloning and Physical Mapping of the
Eco
RI Fragments of the Giant Linear Plasmid SCP1
Authors: Redenbach Matthias , Ikeda Kazuya , Yamasaki Masayuki , Kinashi Haruyasu 
Published: December 2019

ABSTRACT

A cosmid library was constructed for the 350-kb giant linear plasmid SCP1 and aligned on a successive linear map. Only a 0.8-kb gap has remained uncloned in the terminal inverted repeats close to both ends. Partial digestion of the aligned cosmids with
Eco
RI and hybridization with the flanking fragments of the vector enabled physical mapping of all of the
Eco
RI fragments. On this map, the methylenomycin biosynthetic gene cluster, the insertion sequence IS
466
, and the
sapCDE
genes coding for spore-associated proteins were localized.
Nonnative Proteins Induce Expression of theBacillus subtilisCIRCE Regulon
Authors: Mogk Axel , Völker Andrea , Engelmann Susanne , Hecker Michael , Schumann Wolfgang , Völker Uwe 
Published: December 2019

ABSTRACTThe chaperone-encodinggroESLanddnaKoperons constitute the CIRCE regulon ofBacillus subtilis. Both operons are under negative control of the repressor protein HrcA, which interacts with the CIRCE operator and whose activity is modulated by the GroESL chaperone machine. In this report, we demonstrate that induction of the CIRCE regulon can also be accomplished by ethanol stress and puromycin. Introduction of thehrcAgene and a transcriptional fusion under the control of the CIRCE operator intoEscherichia coliallowed induction of this fusion by heat shock, ethanol stress, and overproduction of GroESL substrates. The expression level of thishrcA-bgaBfusion inversely correlated with the amount of GroE machinery present in the cells. Therefore, all inducing conditions seem to lead to induction via titration of the GroE chaperonins by the increased level of nonnative proteins formed. Puromycin treatment failed to induce the ςB-dependent general stress regulon, indicating that nonnative proteins in general do not trigger this response. Reconstitution of HrcA-dependent heat shock regulation ofB. subtilisinE. coliand complementation ofE. coli groESLmutants byB. subtilis groESLindicate that the GroE chaperonin systems of the two bacterial species are functionally exchangeable.
Role of the Multidrug Efflux Systems of
Pseudomonas aeruginosa
in Organic Solvent Tolerance
Authors: Li Xian-Zhi , Zhang Li , Poole Keith 
Published: December 2019

ABSTRACT

Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in
Pseudomonas aeruginosa
. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as
n
-hexane and
p
-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of
P. aeruginosa
. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of
P. aeruginosa
to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.
Determining the Optimal Thymidine Concentration for Growing Thy
−
Escherichia coli
Strains
Authors: Molina Felipe , Jiménez-Sánchez Alfonso , Guzmán Elena C. 
Published: December 2019

ABSTRACT

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy
−
strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy
−
Escherichia coli
strain. Using this method, we found that the
E. coli
“wild-type” strain MG1655 has a partial Thy
−
phenotype.
Multidrug Resistance following Expression of the
Escherichia coli marA
Gene in
Mycobacterium smegmatis
Authors: McDermott Patrick F. , White David G. , Podglajen Isabelle , Alekshun Michael N. , Levy Stuart B. 
Published: December 2019

ABSTRACT

Expression of the
Escherichia coli
multiple antibiotic resistance
marA
gene cloned in
Mycobacterium smegmatis
produced increased resistance to multiple antimicrobial agents, including rifampin, isoniazid, ethambutol, tetracycline, and chloramphenicol. Cloned
marR
or
marA
cloned in the antisense direction had no effect. Resistance changes were lost with spontaneous loss of the plasmid bearing
marA
. A MarA mutant protein, having an insertional mutation within either of its two alpha-helices of the first putative helix-turn-helix domain, failed to produce the multiresistance phenotype in
E. coli
and
M. smegmatis
, indicating that this region is critical for MarA function. These results strongly suggest that
E. coli marA
functions in
M. smegmatis
and that a
mar
-like regulatory system exists in this organism.
Acyl Coenzyme A Synthetase from
Pseudomonas fragi
Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates
Authors: Fontes Rui , Günther Sillero Maria A. , Sillero Antonio 
Published: December 2019

ABSTRACT

Acyl coenzyme A (CoA) synthetase (EC
6.2.1.8
) from
Pseudomonas fragi
catalyzes the synthesis of adenosine 5′-tetraphosphate (p
4
A) and adenosine 5′-pentaphosphate (p
5
A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-
O
-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap
4
A), and adenosine(5′)pentaphospho(5′)adenosine (Ap
5
A) are also substrates of the reaction yielding p
4
(d)A in the presence of tripolyphosphate (P
3
). UTP, CTP, and AMP are not substrates of the reaction. The

K
m

values for ATP and P
3
are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl
2
or CoCl
2
equimolecular with the sum of ATP and P
3
. The relative rates of synthesis of p
4
A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p
4
A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p
4
A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap
4
A (from ATP), Ap
5
A (from p
4
A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap
4
N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).
Characterization of thehcnABCGene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol AgentPseudomonas fluorescensCHA0
Authors: Laville Jacques , Blumer Caroline , Von Schroetter Christine , Gaia Valeria , Défago Geneviève , Keel Christoph , Haas Dieter 
Published: December 2019

ABSTRACTThe secondary metabolite hydrogen cyanide (HCN) is produced byPseudomonas fluorescensfrom glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis inP. fluorescensCHA0 was investigated. The contiguous structural geneshcnABCencoding HCN synthase were expressed from the T7 promoter inEscherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of thehcnABCgenes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. ThehcnApromoter was mapped by primer extension; the −40 sequence (TTGGC … .ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT … .ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned fromP. fluorescensCHA0 and sequenced. ANR of strain CHA0 was most similar to ANR ofP. aeruginosaand CydR ofAzotobacter vinelandii. Ananrmutant ofP. fluorescens(CHA21) produced little HCN and was unable to express anhcnA-lacZtranslational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of thehcnA-lacZfusion. Mutant CHA21 as well as anhcndeletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused byThielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where theanrmutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.