bluuebunny/crossref_metadata_2025 huggingface dataset:
Forespore Expression and Processing of the SigE Transcription Factor in Wild-Type and Mutant
Bacillus subtilis
Authors: Ju Jingliang , Luo Tingqiu , Haldenwang W. G.
Published: December 2019

ς
E
is a mother cell-specific transcription factor of sporulating
Bacillus subtilis
that is derived from an inactive precursor protein (pro-ς
E
). To examine the process that prevents ς
E
activity from developing in the forespore, we fused the ς
E
structural gene (
sigE
) to forespore-specific promoters (

P
dacF

and

P
spoIIIG

), placed these fusions at sites on the
B. subtilis
chromosome which translocate into the forespore either early or late, and used Western blot analysis to monitor SigE accumulation and pro-ς
E
processing.
sigE
alleles, placed at sites which entered the forespore early, were found to generate more protein product than the same fusion placed at a late entering site. SigE accumulation and processing in the forespore were enhanced by null mutations in
spoIIIE
, a gene whose product is essential for translocation of the distal portion of the
B. subtilis
chromosome into the forespore. In other experiments, a chimera of pro-ς
E
and green fluorescence protein, previously shown to be unprocessed if it is synthesized within the forespore, was found to be processed in this compartment if coexpressed with the gene for the pro-ς
E
-processing enzyme, SpoIIGA. The need for
spoIIGA
coexpression is obviated in the absence of SpoIIIE. We interpret these results as evidence that selective degradation of both SigE and SpoIIGA prevent mature ς
E
from accumulating in the forespore compartment of wild-type
B. subtilis
. Presumably, a gene(s) located at a site that is distal to the origin of chromosome transfer is responsible for this phenomenon when it is translocated and expressed in the forespore.
The
Rhizobium etli rpoN
Locus: DNA Sequence Analysis and Phenotypical Characterization of
rpoN
,
ptsN
, and
ptsA
Mutants
Authors: Michiels Jan , Van Soom Tom , D’hooghe Inge , Dombrecht Bruno , Benhassine Traki , de Wilde Petra , Vanderleyden Jos
Published: December 2019

The
rpoN
region of
Rhizobium etli
was isolated by using the
Bradyrhizobium japonicum rpoN1
gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258,
rpoN
, ORF191, and
ptsN
, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and
ptsN
are homologous to conserved ORFs found downstream from
rpoN
genes in other bacterial species. Unlike in most other microorganisms,
rpoN
and ORF191 are separated by approximately 1.6 kb. The
R. etli rpoN
gene was shown to control in free-living conditions the production of melanin, the activation of
nifH
, and the metabolism of C
4
-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the
rpoN
gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the
ptsN
gene resulted in a decrease of melanin synthesis and
nifH
expression. In a search for additional genes controlling the synthesis of melanin, an
R. etli
mutant carrying a Tn
5
insertion in
ptsA
, a gene homologous to the
Escherichia coli
gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The
R. etli ptsA
mutant also displayed reduced expression of
nifH
. The
ptsN
and
ptsA
mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C
4
-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that
ptsN
and
ptsA
are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.
Functions Encoded by Pyrrolnitrin Biosynthetic Genes from
Pseudomonas fluorescens
Authors: Kirner Sabine , Hammer Philip E. , Hill D. Steven , Altmann Annett , Fischer Ilona , Weislo Laura J. , Lanahan Mike , van Pée Karl-Heinz , Ligon James M.
Published: December 2019

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes,
prnABCD
, from
Pseudomonas fluorescens
that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each
prn
gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type
Pseudomonas fluorescens
, but absent from a mutant from which the entire
prn
gene region had been deleted. The
prnA
gene product catalyzes the chlorination of
l
-tryptophan to form 7-chloro-
l
-tryptophan. The
prnB
gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-
l
-tryptophan to monodechloroaminopyrrolnitrin. The
prnC
gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The
prnD
gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the
prn
genes in the operon is identical to the order of the reactions in the biosynthetic pathway.
Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in
Ralstonia eutropha
Authors: Slater Steven , Houmiel Kathryn L. , Tran Minhtien , Mitsky Timothy A. , Taylor Nancy B. , Padgette Stephen R. , Gruys Kenneth J.
Published: December 2019

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism’s intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes.
Ralstonia eutropha
produces both the homopolymer poly-β-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form β-ketovaleryl-CoA. This activity has generally been attributed to the β-ketothiolase encoded by
R. eutropha phbA
. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of
bktB
, which encodes a β-ketothiolase from
R. eutropha
that is capable of forming β-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of β-hydroxyvalerate derived from propionyl-CoA. We also report an additional β-ketothiolase, designated BktC, that probably serves as a secondary route toward β-hydroxyvalerate production.
A Period-Extender Gene,
pex
, That Extends the Period of the Circadian Clock in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Kutsuna Shinsuke , Kondo Takao , Aoki Setsuyuki , Ishiura Masahiro
Published: December 2019

We cloned the pS1K1 plasmid in the process of apparently “complementing” a circadian clock mutant of cyanobacterium
Synechococcus
sp. strain PCC 7942, SP22, which has a 22-h period (T. Kondo, N. F. Tsinoremas, S. S. Golden, C. H. Johnson, S. Kutsuna, and M. Ishiura, Science 266:1233–1236, 1994). Sequence analysis revealed that SP22 did not have a mutation in the genomic DNA segment carried on pS1K1, and the
sp22
mutation was later found in a recently cloned new clock gene,
kaiC
. Therefore, the period-extender gene
pex
that was carried on pS1K1 was a suppressor gene for the
sp22
mutation. The
pex
gene encoded a protein of 148 amino acid residues. No meaningful homologs were found in DNA or protein databases including the
Synechocystis
genome database. The
pex
gene was transcribed from 129 and 164 bp upstream of the translation initiation codon as 0.6-kb transcripts. The Pex protein was detected as a fusion protein with a molecular mass of 15 kDa by the epitope tag fusion method using a c-Myc epitope tag. Disruption of the
pex
gene in wild-type cells shortened the period of the rhythms by 1 h, although it did not affect other properties of the rhythms, whereas its overexpression extended the period by 3 h with a concomitant reduction in the amplitude of the rhythms. In various clock mutants examined, overexpression caused arrhythmicity. Thus, Pex is likely to function as a modifier of the circadian clock in
Synechococcus.
Inorganic Polyphosphate in
Escherichia coli
: the Phosphate Regulon and the Stringent Response
Authors: Rao Narayana N. , Liu Shengjiang , Kornberg Arthur
Published: December 2019

Escherichia coli
transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P
i
), in media deficient in both P
i
and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a
phoB
-containing plasmid,
phoB
mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P
i
starvation). Hence, accumulation of polyP requires a functional
phoB
gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.
The
Bacillus subtilis galE
Gene Is Essential in the Presence of Glucose and Galactose
Authors: Krispin Oliver , Allmansberger Rudolf
Published: December 2019

Bacillus subtilis
is unable to grow by consuming galactose because it is unable to transport it into the cell. The transcription of
galE
is not influenced by galactose but is repressed by glucose. Galactose is toxic for
galE
-negative bacteria because it results in elevated levels of metabolic intermediates. These negative effects are reduced in
galK
and
galT
mutants. Glucose is also toxic for
galE
-negative strains.
Mutational Analysis of the
Chlamydia trachomatis
rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro
Authors: Tan Ming , Gaal Tamas , Gourse Richard L. , Engel Joanne N.
Published: December 2019

We have characterized the
Chlamydia trachomatis
ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified
C. trachomatis
RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous to
Escherichia coli
promoters recognized by E-ς
70
. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for
C. trachomatis
RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by
C. trachomatis
and
E. coli
RNA polymerases. We propose that this region functions as an UP element.
Cloning and Characterization of a Gene Encoding the Major Surface Protein of the Bacterial EndosymbiontWolbachia pipientis
Authors: Braig Henk R. , Zhou Weiguo , Dobson Stephen L. , O’Neill Scott L.
Published: December 2019

The maternally inherited intracellular symbiontWolbachia pipientisis well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressedWolbachiaproteins from infectedDrosophilaeggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of thewsppromoter region was also successfully tested inEscherichia coli. Comparison of sequences of this gene from different strains ofWolbachiarevealed a high level of variability. This sequence variation correlated with the ability of certainWolbachiastrains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool forWolbachiastrain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction ofWolbachiawith its host.
The
Streptomyces peucetius dpsY
and
dnrX
Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis
Authors: Lomovskaya Natalia , Doi-Katayama Yukiko , Filippini Sylvia , Nastro Cecilia , Fonstein Leonid , Gallo Mark , Colombo Anna Luisa , Hutchinson C. Richard
Published: December 2019

The
Streptomyces peucetius dpsY
and
dnrX
genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of
dpsY
and
dnrX
. Replacement of
dpsY
with a mutant form disrupted by insertion of the
aphII
neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of
dnrX
in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that
dnrX
, analogous to the role of the
S. peucetius dnrH
gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.
Promoter Selectivity of the
Bradyrhizobium japonicum
RpoH Transcription Factors In Vivo and In Vitro
Authors: Narberhaus Franz , Kowarik Michael , Beck Christoph , Hennecke Hauke
Published: December 2019

Expression of the
dnaKJ
and
groESL
1
heat shock operons of
Bradyrhizobium japonicum
depends on a ς
32
-like transcription factor. Three such factors (RpoH
1
, RpoH
2
, and RpoH
3
) have previously been identified in this organism. We report here that they direct transcription from some but not all ς
32
-type promoters when the respective
rpoH
genes are expressed in
Escherichia coli
. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH
3
was insoluble. The purified proteins were recognized by an anti-
E. coli
ς
32
serum. While RpoH
1
and RpoH
2
productively interacted with
E. coli
core RNA polymerase and produced
E. coli groE
transcript in vitro, RpoH
3
was unable to do so.
B. japonicum
core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH
1
and RpoH
2
were active, and they initiated transcription at the
B. japonicum groESL
1
and
dnaKJ
promoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the
B. japonicum dnaKJ
and
groESL
1
promoters were suboptimal for transcription by RpoH
1
- or RpoH
2
-containing RNA polymerase from
B. japonicum
. In a mixture of different templates, the
E. coli groESL
promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward
B. japonicum rpoH
-dependent promoters. We conclude that the primary function of RpoH
2
is to supply the cell with DnaKJ under normal growth conditions whereas RpoH
1
is responsible mainly for increasing the level of GroESL
1
after a heat shock.
NADH Dehydrogenase Defects Confer Isoniazid Resistance and Conditional Lethality in
Mycobacterium smegmatis
Authors: Miesel Lynn , Weisbrod Torin R. , Marcinkeviciene Jovita A. , Bittman Robert , Jacobs William R.
Published: December 2019

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of
Mycobacterium tuberculosis
infections. Resistance to INH in clinical isolates has been correlated with mutations in the
inhA
,
katG
, and
ahpC
genes. In this report, we describe a new mechanism for INH resistance in
Mycobacterium smegmatis
. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the
M. tuberculosis
complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD
+
ratio confers INH resistance.
Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex
Authors: We˛grzyn Alicja , Herman-Antosiewicz Anna , Taylor Karol , We˛grzyn Grzegorz
Published: December 2019

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the
Escherichia coli
ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the
groE
operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λ
cro
+
, in λ
cro
−
plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λ
cro
ts plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λ
cro
−
plasmid replication control.
Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants
Authors: Nelson Bryn D. , Traxler Beth
Published: December 2019

The maltose transport complex ofEscherichia coliis a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.
Identification of an
Escherichia coli pepA
Homolog and Its Involvement in Suppression of the
algB
Phenotype in Mucoid
Pseudomonas aeruginosa
Authors: Woolwine Samuel C. , Wozniak Daniel J.
Published: December 2019

Strains of
Pseudomonas aeruginosa
isolated from the respiratory tracts of patients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regulator AlgB is required for full transcription of the alginate biosynthetic operon. Repeated attempts to demonstrate a direct interaction between AlgB and the promoter region of
algD
, the first gene in the alginate operon, have thus far been unsuccessful. The possibility that AlgB exerts its effect on
algD
indirectly exists. To identify putative genes under the control of AlgB which affect
algD
transcription, transposon mutagenesis of nonmucoid
algB
derivatives of the mucoid strain FRD1 was employed. Of approximately 3,000 transposon mutants screened, 6 were found to display phenotypes which were mucoid relative to the phenotype of the parental
algB
strain. The phenotypes of these mutants ranged from being only slightly mucoid to being indistinguishable from that of the original FRD1 strain. One of the particularly mucoid transposon mutants was chosen for further study. This strain was found to be disrupted in a previously uncharacterized open reading frame with 56% amino acid identity to PepA of
Escherichia coli
. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bacterial, plant, and animal species. This novel gene has been designated
phpA
(
P. aeruginosa
homolog of
pepA
). The insertional inactivation of
phpA
was found to correlate with the mucoid phenotype and an increase in
algD
transcription in the
algB
strain. Expression of
phpA
from an ectopic chromosomal locus compensated for the transposon insertion in the native
phpA
gene, restoring
algD
transcription to levels similar to those observed in the parental
algB
strain. While
phpA
expression did not appear to be under the control of AlgB at the transcriptional level, this study demonstrates that loss of
phpA
in an
algB
genetic background had a positive effect on alginate expression and, more specifically, on transcription of the alginate biosynthetic operon.
Chromosome Map of
Xanthomonas campestris
pv. campestris 17 with Locations of Genes Involved in Xanthan Gum Synthesis and Yellow Pigmentation
Authors: Tseng Yi-Hsiung , Choy Ka-Tim , Hung Chih-Hsin , Lin Nien-Tsung , Liu Jane-Yu , Lou Chih-Hong , Yang Bih-Ying , Wen Fu-Shyan , Weng Shu-Fen , Wu Jung-Rung
Published: December 2019

No plasmid was detected in
Xanthomonas campestris
pv. campestris 17, a strain of the causative agent of black rot in cruciferous plants isolated in Taiwan. Its chromosome was cut by
Pac
I,
Pme
I, and
Swa
I into five, two, and six fragments, respectively, and a size of 4.8 Mb was estimated by summing the fragment lengths in these digests. Based on the data obtained from partial digestion and Southern hybridization using probes common to pairs of the overlapping fragments or prepared from linking fragments, a circular physical map bearing the
Pac
I,
Pme
I, and
Swa
I sites was constructed for the
X. campestris
pv. campestris 17 chromosome. Locations of eight
eps
loci involved in exopolysaccharide (xanthan gum) synthesis, two
rrn
operons each possessing an unique
I-Ceu
I site, one
pig
cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn
5
(pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Km
r
) genes, which upon insertion introduced additional sites into the chromosome. The
recA
and
tdh
genes, with known sequences, were mapped by tagging with the polylinker-Km
r
segment from Tn
5
(pfm)CmKm. This is the first map for
X. campestris
and would be useful for genetic studies of this and related
Xanthomonas
species.
Sequence Diversity of Flagellin (fliC) Alleles in PathogenicEscherichia coli
Authors: Reid Sean D. , Selander Robert K. , Whittam Thomas S.
Published: December 2019

To study the molecular evolution of flagellin, the protein subunit specifying flagellar (H) antigens, thefliCgenes from 15 pathogenic strains ofEscherichia coliwere amplified by PCR and sequenced. Comparison offliCsequences of H6 and H7 strains revealed that alleles have a mosaic structure indicating the occurrence of past horizontal transfer of DNA segments between strains. The close similarity of H7 sequences also indicates the exchange of an entirefliCH7 allele between distant clonal lineages. In addition, the ratio of silent substitutions to amino acid replacements suggests that a short segment in the central region offliChas been under positive selection in the divergence of H6 and H7 alleles. Phylogenetic analysis demonstrates that thefliCsequences of O157:H7 and O55:H7 serotypes are nearly identical and highly divergent from those ofE. colistrains expressing H6 and H2 flagellar antigens. A nonmotile clone of sorbitol-fermenting O157 has rapidly accumulated multiple mutations infliC, presumably as a result of the silencing of flagellin expression.
Recruitment of ZipA to the Septal Ring of
Escherichia coli
Is Dependent on FtsZ and Independent of FtsA
Authors: Hale Cynthia A. , de Boer Piet A. J.
Published: December 2019

Cell division in prokaryotes is mediated by the septal ring. In
Escherichia coli
, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.
An Endoglucanase, EglA, from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Hydrolyzes β-1,4 Bonds in Mixed-Linkage (1→3),(1→4)-β-
d
-Glucans and Cellulose
Authors: Bauer Michael W. , Driskill Lance E. , Callen Walter , Snead Marjory A. , Mathur Eric J. , Kelly Robert M.
Published: December 2019

The
eglA
gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon
Pyrococcus furiosus
, was cloned and expressed in
Escherichia coli
. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The
P. furiosus
endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and
p
-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.
SSB
, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes
Authors: Lopez Nelson , Halladay John , Walter William , Craig Elizabeth A.
Published: December 2019

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a
Saccharomyces cerevisiae
Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of
SSB
under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele,
EXA3-1
, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the
EXA3-1
mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of
SSB
, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of
SSB
and ribosomal mRNA after a temperature upshift.
Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo
Authors: Dekker Niek , Tommassen Jan , Verheij Hubertus M.
Published: December 2019

Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin from
Escherichia coli
. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.
The Type II Pullulanase of
Thermococcus hydrothermalis
: Molecular Characterization of the Gene and Expression of the Catalytic Domain
Authors: Erra-Pujada Marta , Debeire Philippe , Duchiron Francis , O’Donohue Michael J.
Published: December 2019

The gene encoding a hyperthermostable type II pullulanase produced by
Thermococcus hydrothermalis
(Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (
apuA
) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.
Regulation of β-Galactosidase Expression in
Bacillus megaterium
DSM319 by a XylS/AraC-Type Transcriptional Activator
Authors: Strey Jan , Wittchen Klaus D. , Meinhardt Friedhelm
Published: December 2019

The β-galactosidase-encoding
bgaM
gene of
Bacillus megaterium
DSM319 and the divergently orientated
bgaR
operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of
bgaM
; a potential regulator binding site overlaps the
bgaM
promoter. A mechanism for regulation of β-galactosidase expression in
B. megaterium
is proposed.
SufS Is a NifS-Like Protein, and SufD Is Necessary for Stability of the [2Fe-2S] FhuF Protein in
Escherichia coli
Authors: Patzer Silke I. , Hantke Klaus
Published: December 2019

Escherichia coli fhuF
mutants, a
sufS
::MudI mutant, and a
sufD
::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the
sufS
and
sufD
genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the
sufS
open reading frame corresponds to
orf f406
. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream
sufD
gene (
orf f423
) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a Δ
sufD
strain failed. Radioactive labeling of N-His-FhuF with [
35
S]methionine showed that the protein was unstable in the Δ
sufD
mutant.
A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-
p
-Dioxin Pathway Genes in
Sphingomonas
sp. Strain RW1
Authors: Armengaud Jean , Timmis Kenneth N. , Wittich Rolf-Michael
Published: December 2019

The bacterium
Sphingomonas
sp. strain RW1 is able to use dibenzo-
p
-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp
Hin
dIII fragment downstream of cistrons
dxnA1A2
, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in
Escherichia coli
. The first three ORFs of the locus, designated
dxnC
, ORF2, and
fdx3
, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively.
dxnD
encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and
dxnE
codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene,
dxnF
, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-
cis,cis
-muconate. The heteromeric protein encoded by
dxnGH
is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas
dxnI
specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-
p
-dioxin in
Sphingomonas
sp. strain RW1 thus suggests itself.
Cells of
Escherichia coli
Contain a Protein-Tyrosine Kinase, Wzc, and a Phosphotyrosine-Protein Phosphatase, Wzb
Authors: Vincent Carole , Doublet Patricia , Grangeasse Christophe , Vaganay Elisabeth , Cozzone Alain J. , Duclos Bertrand
Published: December 2019

Two proteins of
Escherichia coli
, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate
p
-nitrophenyl phosphate into
p
-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that
E. coli
cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.
Control of Acid Resistance in
Escherichia coli
Authors: Castanie-Cornet Marie-Pierre , Penfound Thomas A. , Smith Dean , Elliott John F. , Foster John W.
Published: December 2019

Acid resistance (AR) in
Escherichia coli
is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in
E. coli
. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (
adiA
), and the regulator
cysB
, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (
gadA
or
gadB
), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (
gadC
). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the
gad
genes proved separable. Stationary-phase induction of
gadA
and
gadB
required the alternative sigma factor ς
S
encoded by
rpoS
. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ς
S
independent. Neither
gad
gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ς
S
-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus,
E. coli
possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of
Escherichia coli
, ς
32
Authors: Arsène Florence , Tomoyasu Toshifumi , Mogk Axel , Schirra Christiane , Schulze-Specking Agnes , Bukau Bernd
Published: December 2019

Expression of heat shock genes is controlled in
Escherichia coli
by the antagonistic action of the ς
32
subunit of RNA polymerase and the DnaK chaperone system, which inactivates ς
32
by stress-dependent association and mediates ς
32
degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among ς
32
homologs, termed region C, was proposed to play a role in ς
32
degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of ς
32
, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the ς
32
protein did not affect ς
32
degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in ς
32
control by DnaK and FtsH. Instead, the ς
32
mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of ς
32
to RNA polymerase. Region C thus confers on ς
32
a competitive advantage over other ς factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.
The
mdoC
Gene of
Escherichia coli
Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans
Authors: Lacroix Jean-Marie , Lanfroy Eric , Cogez Virginie , Lequette Yannick , Bohin Anne , Bohin Jean-Pierre
Published: December 2019

Osmoregulated periplasmic glucans (OPGs) of
Escherichia coli
are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient
mdoB
mutant was subjected to Tn
5
transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named
mdoC
.
mdoC
, which encodes a membrane-bound protein, is closely linked to the
mdoGH
operon necessary for the synthesis of the OPG backbone.
relA
Is Required for Actinomycin Production in
Streptomyces antibioticus
Authors: Hoyt Shannan , Jones George H.
Published: December 2019

The
relA
gene from
Streptomyces antibioticus
has been cloned and sequenced. The gene encodes a protein with an
M
r
of 93,653, which is 91% identical to the corresponding protein from
Streptomyces coelicolor
. Disruption of
S. antibioticus relA
produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact
relA
gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted
relA
gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the
relA
disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.
The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in
Erwinia chrysanthemi
3937
Authors: Shevchik Vladimir E. , Condemine Guy , Robert-Baudouy Janine , Hugouvieux-Cotte-Pattat Nicole
Published: December 2019

Erwinia chrysanthemi
3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of
E. chrysanthemi
. The
E. chrysanthemi
3937 gene
kdgC
has been shown to exhibit a high degree of similarity to the genes
pelY
of
Yersinia pseudotuberculosis
and
pelB
of
Erwinia carotovora
, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the
kdgC
gene was renamed
pelW
. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca
2+
is unable to restore the activity of PelW or Ogl, while several other cations, including Co
2+
, Mn
2+
, and Ni
2+
, can activate both cytoplasmic lyases.