bluuebunny/crossref_metadata_2025 huggingface dataset:
The 102-KilobasepgmLocus ofYersinia pestis: Sequence Analysis and Comparison of Selected Regions among DifferentYersinia pestisandYersinia pseudotuberculosisStrains
Authors: Buchrieser Carmen , Rusniok Christophe , Frangeul Lionel , Couve Elisabeth , Billault Alain , Kunst Frank , Carniel Elisabeth , Glaser Philippe
Published: December 2019

We report the complete 119,443-bp sequence of thepgmlocus fromYersinia pestisand its flanking regions. Sequence analysis confirms that the 102-kb unstablepgmlocus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system ofBordetella pertussis. Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among differentY. pestisandYersinia pseudotuberculosisstrains, the sequence of selected regions of thepgmlocus and flanking regions were compared from 20 differentY. pestisand 10Y. pseudotuberculosisstrains. The results showed that the genes interrupted inY. pestisare intact inY. pseudotuberculosis. However, one of these mutations, in thebvgShomologue, is only present inY. pestisstrains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29Yersiniastrains suggest thatY. pestisis a recently emerged pathogen that is probably entering the initial phase of reductive evolution.
Control of Filament Formation in
Candida albicans
by Polyamine Levels
Authors: Herrero Ana B. , López M. Carmen , García Susana , Schmidt Axel , Spaltmann Frank , Ruiz-Herrera José , Dominguez Angel
Published: December 2019

Candida albicans
, the most common fungal pathogen, regulates its cellular morphology in response to environmental conditions. The
ODC
gene, which encodes ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was isolated and disrupted. Homozygous null
Candida
mutants behaved as polyamine auxotrophs and grew exclusively in the yeast form at low polyamine levels (0.01 mM putrescine) under all conditions tested. An increase in the polyamine concentration (10 mM putrescine) restored the capacity to switch from the yeast to the filamentous form. The strain with a deletion mutation also showed increased sensitivity to salts and calcofluor white. This
Candida odc/odc
mutant was virulent in a mouse model. The results suggest a model in which polyamine levels exert a pleiotrophic effect on transcriptional activity.
Dynamics of Iron Uptake and Fe3O4Biomineralization during Aerobic and Microaerobic Growth ofMagnetospirillum gryphiswaldense
Authors: Schüler Dirk , Baeuerlein Edmund
Published: December 2019

Iron uptake and magnetite (Fe3O4) crystal formation could be studied in the microaerophilic magnetic bacteriumMagnetospirillum gryphiswaldenseby using a radioactive tracer method for iron transport and a differential light-scattering technique for magnetism. Magnetite formation occurred only in a narrow range of low oxygen concentration, i.e., 2 to 7 μM O2at 30°C. Magnetic cells stored up to 2% iron as magnetite crystals in intracytoplasmic vesicles. This extraordinary uptake of iron was coupled tightly to the biomineralization of up to 60 magnetite crystals with diameters of 42 to 45 nm.
Identification and Characterization of a New Organic Hydroperoxide Resistance (
ohr
) Gene with a Novel Pattern of Oxidative Stress Regulation from
Xanthomonas campestris
pv. phaseoli
Authors: Mongkolsuk Skorn , Praituan Wipa , Loprasert Suvit , Fuangthong Mayuree , Chamnongpol Sangpen
Published: December 2019

We have isolated a new organic hydroperoxide resistance (
ohr
) gene from
Xanthomonas campestris
pv. phaseoli. This was done by complementation of an
Escherichia coli
alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype.
ohr
encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An
ohr
mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H
2
O
2
or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant.
ohr
had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H
2
O
2
, and not induced at all by a superoxide generator. The novel regulation pattern of
ohr
suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of
ohr
in various bacteria tested conferred increased resistance to
tert
-butyl hydroperoxide killing, but this was not so for wild-type
Xanthomonas
strains. The organic hydroperoxide hypersensitivity of
ohr
mutants could be fully complemented by expression of
ohr
or a combination of
ahpC
and
ahpF
and could be partially complemented by expression
ahpC
alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.
Isolation and Characterization of
Methanobacterium thermoautotrophicum
ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions
Authors: Pennings Jeroen L. A. , Keltjens Jan T. , Vogels Godfried D.
Published: December 2019

By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of
Methanobacterium thermoautotrophicum
that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F
420
-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F
390
, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.
Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium
Rhodothermus obamensis
Authors: Takai Ken , Nunoura Takuro , Sako Yoshihiko , Uchida Aritsune
Published: December 2019

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium
Rhodothermus obamensis
. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.
Paradoxical Enhancement of the Activity of a Bacterial Multidrug Transporter Caused by Substitutions of a Conserved Residue
Authors: Klyachko Katya A. , Neyfakh Alexander A.
Published: December 2019

Substitution of threonine or serine for the evolutionary conserved intramembrane proline P
347
of the
Bacillus subtilis
multidrug transporter Bmr significantly increases the toxin-effluxing activity of Bmr without affecting its abundance in the cell. In cocultivation experiments, we demonstrate that although the mutant T
347
Bmr is advantageous to cells growing in the presence of a toxin, the wild-type P
347
Bmr is advantageous under the conditions of nutritional limitation. This may explain why Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution of Bmr has been determined by its presently unidentified natural function rather than by its ability to expel diverse toxins from the cell.
Molecular Characterization of the Gene Encoding the DNA Gyrase A Subunit of
Streptococcus pneumoniae
Authors: Balas Delia , Fernández-Moreira Esteban , De La Campa Adela G.
Published: December 2019

The gene encoding the DNA gyrase A subunit of
Streptococcus pneumoniae
was cloned and sequenced. The
gyrA
gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an
Escherichia coli
expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of
gyrA
. The bend center was mapped and located between the −35 and −10 regions of the promoter. Primer extension analysis showed that
gyrA
transcription initiates 6 bp downstream of an extended −10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of
gyrA
are discussed.
Characterization of the
hca
Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in
Escherichia coli
K-12
Authors: Díaz Eduardo , Ferrández Abel , García José L.
Published: December 2019

We have identified, cloned, and sequenced the
hca
cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in
Escherichia coli
K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (
hcaA1A2CBD
) and two additional genes transcribed in the opposite direction that encode a potential permease (
hcaT
) and a regulator (
hcaR
). Sequence comparisons revealed that while
hcaA1A2CD
genes encode the four subunits of the 3-phenylpropionate dioxygenase, the
hcaB
gene codes for the corresponding
cis
-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in
E. coli
. The inducible expression of the
hca
genes requires the presence of the
hcaR
gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy derivative. Further catabolism of DHPP requires the
mhp
-encoded
meta
fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, J. Bacteriol. 179:2573–2581, 1997). Expression in
Salmonella typhimurium
of the
mhp
genes alone or in combination with the
hca
cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent
mhp
- and
hca
-encoded pathways are also functional in
S. typhimurium
, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.
In Vitro Synthesis of Multicopy Single-Stranded DNA, Using Separate Primer and Template RNAs, by
Escherichia coli
Reverse Transcriptase
Authors: Shimamoto Tadashi , Kawanishi Hideki , Tsuchiya Tomofusa , Inouye Sumiko , Inouye Masayori
Published: December 2019

A minor population of wild strains of
Escherichia coli
contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the
msr-msd
region under a single promoter. A single RNA transcript from the
msr-msd
region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed
msr
and
msd
transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5′-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2′-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the
msr
RNA transcript.
An Isoflavonoid-Inducible Efflux Pump in
Agrobacterium tumefaciens
Is Involved in Competitive Colonization of Roots
Authors: Palumbo Jeffrey D. , Kado Clarence I. , Phillips Donald A.
Published: December 2019

Agrobacterium tumefaciens
1D1609, which was originally isolated from alfalfa (
Medicago sativa
L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn
5
-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame,
ifeA
, of a three-gene locus (
ifeA
,
ifeB
, and
ifeR
), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (
P
≤ 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (
ifeA
::
gusA
) and I-6 (
ifeA
::Ω-Tc), confirmed the importance of
ifeA
for competitive root colonization. Exposure to the isoflavonoid coumestrol increased β-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the
ifeA-gusA
reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on
A. tumefaciens
in an environment where the inducing molecules are known to be present.
Effects of Carbon Source on Expression of F
o
Genes and on the Stoichiometry of the c Subunit in the F
1
F
o
ATPase of
Escherichia coli
Authors: Schemidt Randy A. , Qu Jun , Williams James R. , Brusilow William S. A.
Published: December 2019

Expression of the genes for the membrane-bound F
o
sector of the
Escherichia coli
F
1
F
o
proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F
o
proton channel. Transcriptional and translational
lacZ
fusions to the promoter and to two F
o
genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of
uncB
, encoding the a subunit of F
o
, are higher than during growth on glucose. In contrast, translation of the
uncE
gene, encoding the c subunit of F
o
, is higher during growth on glucose than during growth on succinate. Translation rates of both
uncB
and
uncE
change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F
1
F
o
ATPase in cells grown on glucose than in cells grown on succinate.
E. coli
therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.
The
MTCY428.08
Gene of
Mycobacterium tuberculosis
Codes for NAD
+
Synthetase
Authors: Cantoni Rita , Branzoni Manuela , Labò Monica , Rizzi Menico , Riccardi Giovanna
Published: December 2019

The product of the
MTCY428.08
gene of
Mycobacterium tuberculosis
shows sequence homology with several NAD
+
synthetases. The
MTCY428.08
gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in
Escherichia coli
led to overproduction of glutathione
S
-transferase fused and His
6
-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the
MTCY428.08
gene of
M. tuberculosis
is the structural gene for NAD
+
synthetase.
Anaerobic Regulation of
Bacillus subtilis
Krebs Cycle Genes
Authors: Nakano Michiko M. , Zuber Peter , Sonenshein Abraham L.
Published: December 2019

Krebs cycle enzyme activity in
Bacillus subtilis
was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions. The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures. These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription. This repression was not dependent on either the
fnr
or
resDE
gene products, which have been shown to regulate expression of other
B. subtilis
genes in response to anaerobic conditions. Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression. A dyad symmetry element located between positions −73 and −59 relative to the transcription start site of the aconitase gene (
citB
) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth. The deletion of the upstream arm of the dyad symmetry region abolished the
citB
repression observed during anaerobic growth. Furthermore, neither
citZ
or
citB
was repressed in an anaerobically grown
citB
mutant, an effect that was very likely the result of citrate accumulation. These results suggest that catabolite repression and anaerobic repression of
citZ
and
citB
are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.
Aeromonas hydrophila
Adenylyl Cyclase 2: a New Class of Adenylyl Cyclases with Thermophilic Properties and Sequence Similarities to Proteins from Hyperthermophilic Archaebacteria
Authors: Sismeiro Odile , Trotot Pascale , Biville Francis , Vivares Christian , Danchin Antoine
Published: December 2019

Complementation of an
Escherichia coli cya
mutant with a genomic library from
Aeromonas hydrophila
allowed isolation of clones containing two different
cya
genes. Whereas one of these genes (
cyaA
) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (
cyaB
) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65°C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in
A. hydrophila
, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to
cyaA
, the
cyaB
gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the
cyaB
gene in a
cyaA
mutant, as well as in a
cyaA cyaB
mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria:
Methanobacterium thermoautotrophicum
,
Archaeglobus fulgidus
, and
Methanococcus jannaschii
. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.
Copper-Binding Compounds from
Methylosinus trichosporium
OB3b
Authors: DiSpirito Alan A. , Zahn James A. , Graham David W. , Kim Hyung J. , Larive Cynthia K. , Derrick Tiffany S. , Cox Charles D. , Taylor Alan
Published: December 2019

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO
C
) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of
Methylosinus trichosporium
OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L
1
and CBC-L
2
, respectively. The amino acid sequence of CBC-L
1
is S?MYPGS?M, and that of CBC-L
2
is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L
1
showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g
⊥
= 2.087, g
∥︀
= 2.42 G, ‖A
∥︀
‖ = 128 G). The EPR spectrum of CBC-L
2
was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu
2+
center (g
⊥
= 2.073, g
∥︀
= 2.324 G, ‖A
∥︀
‖ = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g
⊥
= 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO
C
mutants PP319 and PP359 regardless of the copper concentration in the culture medium.
RpoS (Sigma-S) Controls Expression of
rsmA
, a Global Regulator of Secondary Metabolites, Harpin, and Extracellular Proteins in
Erwinia carotovora
Authors: Mukherjee Asita , Cui Yaya , Ma Weilei , Liu Yang , Ishihama Akira , Eisenstark Abraham , Chatterjee Arun K.
Published: December 2019

RpoS (sigma-S or sigma-38) controls a large array of genes that are expressed during stationary phase and under various stress conditions in
Escherichia coli
and other bacteria. We document here that plant pathogenic and epiphytic
Erwinia
species, such as
E. amylovora
;
E. carotovora
subsp.
atroseptica
,
betavasculorum
, and
carotovora
;
E. chrysanthemi
;
E. herbicola
;
E. rhapontici
; and
E. stewartii
, possess
rpoS
genes and produce the alternate sigma factor. We show that
rpoS
transcription in
E. carotovora
subsp.
carotovora
is driven from a major promoter which resides within the
nlpD
gene located upstream of
rpoS
as in
E. coli
. RpoS
−
E. carotovora
subsp.
carotovoa
strain AC5061, constructed by marker exchange, is more sensitive to hydrogen peroxide, carbon starvation, and acidic pH than its RpoS
+
parent strain, AC5006. The basal levels of extracellular pectate lyase, polygalacturonase, and cellulase as well as those of transcripts of
E. carotovora
subsp.
carotovora hrpN
(

hrpN
Ecc

), the gene for the elicitor of the hypersensitive reaction, are higher in the RpoS
−
strain than in the RpoS
+
parent. Likewise, compared to AC5006, AC5061 causes more extensive maceration of celery petioles. Our findings with the RpoS
−
mutant and strains carrying multiple copies
rpoS
+
DNA reveal that
rpoS
positively controls
rsmA
expression. We also present evidence that supports the hypothesis that the RpoS effect on extracellular enzyme levels,

hrpN
Ecc

expression, and virulence manifests itself by the modulation of
rsmA
expression.
Tca1, the Retrotransposon-Like Element of
Candida albicans
, Is a Degenerate and Inactive Element
Authors: Chen Jiang-ye , Wang Qin , Fu Zheng , Zhou Song , Fonzi William A.
Published: December 2019

Candida albicans
is an asexual fungus and as such must rely on mechanisms other than sexual recombination to generate genetic diversity. Retrotransposons are ubiquitous genetic elements known to generate multiple types of genomic alterations. We have further investigated the nature of the retrotransposon-like element Tca1 in
C. albicans
. Tca1 is present at two loci in strain SC5314. Both loci have now been cloned, and one element was sequenced in its entirety. This element was flanked by α elements, or long terminal repeats (LTRs), and contained an intervening region of 5,614 bp. The intervening region was highly degenerate and contained no extended open reading frames, indicating that Tca1 is not a functional element. Partial sequence determination demonstrated that the elements from the two loci were nearly identical. Genetic manipulation of the elements showed that both loci were heterozygous for Tca1, that both were transcriptionally active, and that deletion of both had no effect on growth rate or germ tube formation. Thus, it is unclear why this nonfunctional, highly degenerate element has been maintained in many clinical isolates.
Sequence Analysis of the GntII (Subsidiary) System for Gluconate Metabolism Reveals a Novel Pathway for
l
-Idonic Acid Catabolism in
Escherichia coli
Authors: Bausch Christoph , Peekhaus Norbert , Utz Cristina , Blais Tessa , Murray Elizabeth , Lowary Todd , Conway Tyrrell
Published: December 2019

The presence of two systems in
Escherichia coli
for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of
l
-idonic acid in which
d
-gluconate is an intermediate. The genes have been named accordingly: the
idnK
gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the
idnD-idnO-idnT-idnR
operon, which encodes
l
-idonate 5-dehydrogenase, 5-keto-
d
-gluconate 5-reductase, an
l
-idonate transporter, and an
l
-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of
l
-idonate; IdnD catalyzes a reversible oxidation of
l
-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form
d
-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of
d
-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with
l
-idonate or 5-ketogluconate serving as the true inducer of the pathway. The
l
-idonate 5-dehydrogenase and 5-keto-
d
-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of
l
-idonate and
d
-gluconate via the intermediate 5-ketogluconate.
E. coli
K-12 strains are able to utilize
l
-idonate as the sole carbon and energy source, and as predicted, the ability of
idnD
,
idnK
,
idnR
, and
edd
mutants to grow on
l
-idonate is altered.
Diversity of Cytochrome
bc
Complexes: Example of the Rieske Protein in Green Sulfur Bacteria
Authors: Brugna Myriam , Albouy Delphine , Nitschke Wolfgang
Published: December 2019

The Rieske 2Fe2S cluster of
Chlorobium limicola
forma
thiosulfatophilum
strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone. A redox midpoint potential (

E
m

) of +160 mV in the pH range of 6 to 7.7 and a decreasing

E
m

(−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its

E
m

-versus-pH curve, in green sulfur bacteria are discussed.
Cofactor Engineering: a Novel Approach to Metabolic Engineering in
Lactococcus lactis
by Controlled Expression of NADH Oxidase
Authors: Lopez de Felipe Felix , Kleerebezem Michiel , de Vos Willem M. , Hugenholtz Jeroen
Published: December 2019

NADH oxidase-overproducing
Lactococcus lactis
strains were constructed by cloning the
Streptococcus mutans nox-2
gene, which encodes the H
2
O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the
L. lactis nisA
promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD
+
ratios in the cells.
A Transcriptional Activator, Homologous to the
Bacillus subtilis
PurR Repressor, Is Required for Expression of Purine Biosynthetic Genes in
Lactococcus lactis
Authors: Kilstrup Mogens , Martinussen Jan
Published: December 2019

A
purR
::pGh9:IS
S1
mutant of
Lactococcus lactis
was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxotrophs. After determination of the nucleotide sequence and deduction of the
purR
reading frame, the PurR product was found to be highly similar to the
purR
-encoded repressor from
Bacillus subtilis
. The wild-type
purR
gene complemented the purine auxotrophy of a
purR
::IS
S1
mutant, and it was shown that the
purR
::IS
S1
mutation lowered the level of transcription from the purine-regulated
L. lactis purD
promoter. In a parallel study on the regulation of
purC
and
purD
expression in
L. lactis
(M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900–3906, 1998), we identified regions (PurBox sequences: AWWWCCGAACWWT) upstream of the promoters with a central G residue at exactly position −76 relative to the transcriptional start site. The PurBox sequences were found to be required for high-level promoter activity and purine regulation. We identified a PurBox sequence overlapping the −35 region of the
L. lactis purR
promoter and found, by studies of a
purR-lacLM
fusion plasmid, that
purR
is autoregulated. Because of the high degree of similarity of the PurR proteins from
B. subtilis
and
L. lactis
, we looked for PurBox sequences in the promoter regions of the PurR-regulated genes in
B. subtilis
and identified a perfectly matching PurBox sequence in the
purA
promoter region and slightly degenerate PurBox-like sequences in the promoter regions for the
pur
operon and the
purR
gene. Interestingly, the PurBox in the
pur
operon of
B. subtilis
is located almost identically, with respect to the promoter, to the PurBox sequences located in front of
purC
and
purD
in
L. lactis
. We present a hypothesis to explain how an ancestral PurR protein in
B. subtilis
could have evolved from an activator of the
pur
operon into a repressor which regulates transcription initiation from the same
pur
promoter by using the same PurR binding site and a similar response toward its effectors.
Functional Expression in
Lactobacillus plantarum
of
xylP
Encoding the Isoprimeverose Transporter of
Lactobacillus pentosus
Authors: Chaillou Stéphane , Postma Pieter W. , Pouwels Peter H.
Published: December 2019

The
xylP
gene of
Lactobacillus pentosus
, the first gene of the
xylPQR
operon, was recently found to be involved in isoprimeverose metabolism. By expression of
xylP
on a multicopy plasmid in
Lactobacillus plantarum
80, a strain which lacks active isoprimeverose and
d
-xylose transport activities, it was shown that
xylP
encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in
L. plantarum
80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of
d
-xylose.
Investigation of theStreptomyces clavuligerusCephamycin C Gene Cluster and Its Regulation by the CcaR Protein
Authors: Alexander Dylan C. , Jensen Susan E.
Published: December 2019

As part of a search for transcriptional regulatory genes, sequence analysis of several previously unsequenced gaps in the cephamycin biosynthetic cluster has revealed the presence inStreptomyces clavuligerusof seven genes not previously described. These include genes encoding an apparent penicillin binding protein and a transport or efflux protein, as well as the CmcI and CmcJ proteins, which catalyze late reactions in the cephamycin biosynthetic pathway. In addition, we discovered a gene, designatedpcd, which displays significant homology to genes encoding semialdehyde dehydrogenases and may represent the gene encoding the long-sought-after dehydrogenase involved in the conversion of lysine to α-aminoadipate. Finally, two genes,sclUandrhsA, with no obvious function in cephamycin biosynthesis may define the end of the cluster. The previously described CcaR protein displays homology to a number ofStreptomycespathway-specific transcriptional activators. TheccaRgene was shown to be essential for the biosynthesis of cephamycin, clavulanic acid, and non-clavulanic acid clavams. Complementation of a deletion mutant lackingccaRand the adjacentorf11andblpgenes showed that onlyccaRwas essential for the biosynthesis of cephamycin, clavulanic acid, and clavams and that mutations inorf11orblphad no discernible effects. The lack of cephamycin production inccaRmutants was directly attributable to the absence of biosynthetic enzymes responsible for the early and middle steps of the cephamycin biosynthetic pathway. Complementation of theccaRdeletion mutant resulted in the return of these biosynthetic enzymes and the restoration of cephamycin production.
Parallel and Divergent Genotypic Evolution in Experimental Populations of
Ralstonia
sp
Authors: Nakatsu Cindy H. , Korona Ryszard , Lenski Richard E. , de Bruijn Frans J. , Marsh Terence L. , Forney Larry J.
Published: December 2019

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from
Ralstonia
sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the
tfdA
gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2.4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.
Chromosomal Integration, Tandem Amplification, and Deamplification in
Pseudomonas putida
F1 of a 105-Kilobase Genetic Element Containing the Chlorocatechol Degradative Genes from
Pseudomonas
sp. Strain B13
Authors: Ravatn Roald , Studer Sonja , Springael Dirk , Zehnder Alexander J. B. , van der Meer Jan Roelof
Published: December 2019

Analysis of chlorobenzene-degrading transconjugants of
Pseudomonas putida
F1 which had acquired the genes for chlorocatechol degradation (
clc
) from
Pseudomonas
sp. strain B13 revealed that the
clc
gene cluster was present on a 105-kb amplifiable genetic element (named the
clc
element). In one such transconjugant,
P. putida
RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb
clc
element occurred in two different loci, and the target sites were located within the 3′ end of glycine tRNA structural genes. Tandem amplification of the
clc
element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the
clc
element. Two nonadjacent copies of the
clc
element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate. This result suggests that the presence of multiple copies of the
clc
gene cluster was a prerequisite for the growth of
P. putida
RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene.
Molecular Cloning and Characterization of
cgs
, the
Brucella abortus
Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of
Rhizobium meliloti ndvB
and
Agrobacterium tumefaciens chvB
Mutants
Authors: Iñón de Iannino Nora , Briones Gabriel , Tolmasky Marcelo , Ugalde Rodolfo A.
Published: December 2019

The animal pathogen
Brucella abortus
contains a gene,
cgs
, that complemented a
Rhizobium meliloti
nodule development (
ndvB
) mutant and an
Agrobacterium tumefaciens
chromosomal virulence (
chvB
) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in
A. tumefaciens
, and nitrogen fixation in
R. meliloti
; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in
B. abortus
of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to
R. meliloti
NdvB. Four regions of the
B. abortus
protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with
R. meliloti
NdvB. Tn
3
-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the
Brucella
protein, although highly conserved in
Rhizobium
, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as
B. abortus
cyclic β(1-2) glucan synthetase was done by the construction of a
B. abortus
Tn
3
-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in
Brucella
infection.
Mutations That Alter the Kinase and Phosphatase Activities of the Two-Component Sensor EnvZ
Authors: Hsing Weihong , Russo Frank D. , Bernd Karen K. , Silhavy Thomas J.
Published: December 2019

EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in
Escherichia coli
in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K
+
P
−
) and the OmpR-P phosphatase-dominant state (K
−
P
+
). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.
Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from
Pseudomonas pseudoalcaligenes
JS45
Authors: He Zhongqi , Davis John K. , Spain Jim C.
Published: December 2019

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by
Pseudomonas pseudoalcaligenes
JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD
+
. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.
Only the N-Terminal Domain of FtsK Functions in Cell Division
Authors: Draper G. Craig , McLennan Neil , Begg Ken , Masters Millicent , Donachie William D.
Published: December 2019

Deletion of
ftsK
results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first ∼17% of
ftsK
. The division block can be suppressed in most mutants by deletion of
dacA
, which codes for the
d
-alanine:
d
-alanine carboxypeptidase PBP5, or in all mutants by overexpression of
ftsN
. Overexpression of
ftsK
inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.
Transcriptional Activation of the
Bacillus subtilis spoIIG
Promoter by the Response Regulator Spo0A Is Independent of the C-Terminal Domain of the RNA Polymerase Alpha Subunit
Authors: Rowe-Magnus Dean A. , Mencía Mario , Rojo Fernando , Salas Margarita , Spiegelman George B.
Published: December 2019

In vitro transcription from the
spoIIG
promoter by
Bacillus subtilis
RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.
A Region in
Bacillus subtilis
ς
H
Required for Spo0A-Dependent Promoter Activity
Authors: Buckner Cindy M. , Moran Charles P.
Published: December 2019

Spo0A activates transcription in
Bacillus subtilis
from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, ς
A
, and RNA polymerase containing a secondary sigma factor, known as ς
H
. The region of ς
A
near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of ς
A
at these promoters. To determine if the amino acids in the corresponding region of ς
H
are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in ς
H
(201 to 210). Two alanine substitutions in ς
H
, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, ς
H
-dependent promoter
spoIIA
but did not affect expression from the ς
H
-dependent, Spo0A-independent promoters
citGp2
and
spoVG
. Therefore, promoter activation by Spo0A requires homologous regions in ς
A
and ς
H
. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in ς
A
did not suppress the sporulation defects caused by the Q201A and R205A substitutions in ς
H
. This result and others indicate that different surfaces of Spo0A probably interact with ς
A
and ς
H
RNA polymerases.