bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning of the Actinomycin Synthetase Gene Cluster from
Streptomyces chrysomallus
and Functional Heterologous Expression of the Gene Encoding Actinomycin Synthetase II
Authors: Schauwecker Florian , Pfennig Frank , Schröder Werner , Keller Ullrich
Published: December 2019

The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (
acmA
) and ACMS II (
acmB
) by hybridization screening of a cosmid library of
Streptomyces chrysomallus
DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (
acmC
), is located immediately downstream of
acmB
in the same orientation. The protein sequence of ACMS II, deduced from
acmB
, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of
acmB
from the
mel
promoter of plasmid PIJ702 in
Streptomyces lividans
yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl–
l
-valine, which is epimerized to threonyl–
d
-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from
S. chrysomallus.
Thioredoxin Is Involved in Oxygen-Regulated Formation of the Photosynthetic Apparatus of
Rhodobacter sphaeroides
Authors: Pasternak Cecile , Haberzettl Kerstin , Klug Gabriele
Published: December 2019

Thioredoxin, a redox active protein, has been previously demonstrated to be essential for growth of the anoxygenic photosynthetic bacterium
Rhodobacter sphaeroides
. In the present study, the involvement of thioredoxin in the formation of the photosynthetic apparatus of
R. sphaeroides
WS8 was investigated by construction and analysis of a mutant strain disrupted for the chromosomal
trxA
copy and carrying a plasmid-borne copy of
trxA
under the control of the hybrid p

trc

promoter inducible by IPTG (isopropyl-β-
d
-thiogalactopyranoside). This strain was viable in the absence of IPTG but was affected in pigmentation. When shifted from high to low oxygen tension conditions, the
trxA
mutant showed a reduced bacteriochlorophyll content in comparison to that of the wild type. Although thioredoxin is able to regulate aminolevulinic acid (ALA) synthase (the first enzyme of the tetrapyrrole biosynthetic pathway) activity by a dithiol-disulfide exchange, our mutant strain exhibited a level of ALA synthase activity identical to that of the wild type, suggesting that thioredoxin is involved in other steps to regulate the synthesis of the photosynthetic apparatus. Accordingly, we showed that the
trxA
mutation affects the oxygen-regulated expression of the
puf
operon encoding the pigment-binding proteins of the light-harvesting and reaction center complexes. Upon transition from aerobic to semiaerobic growth conditions, the maximal
puf
mRNA level was found to be 40 to 50% lower in the mutant strain than in the wild type. The stability of the
puf
transcripts was identical in both strains grown under low oxygen tension, indicating that the role of thioredoxin in regulating
puf
expression occurs at the transcriptional level.
Molecular and Biochemical Characterization of the Protein Template Controlling Biosynthesis of the Lipopeptide Lichenysin
Authors: Konz Dirk , Doekel Sascha , Marahiel Mohamed A.
Published: December 2019

Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains ofBacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon fromB. licheniformisATCC 10716. Three large open reading frames coding for peptide synthetases, designatedlicA,licB(three modules each), andlicC(one module), could be detected, followed by a gene,licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed inEscherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequencel-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.
A Membrane-Bound NAD(P)
+
-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by
Klebsiella pneumoniae
Authors: Steuber Julia , Krebs Walter , Bott Michael , Dimroth Peter
Published: December 2019

During anaerobic growth of
Klebsiella pneumoniae
on citrate, 9.4 mmol of H
2
/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO
2
. Upon addition of NiCl
2
(36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H
2
-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)
+
was also achieved by the addition of formate. In crude extracts, the H
2
:NAD
+
oxidoreductase activity was 0.13 μmol min
−1
mg
−1
, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min
−1
mg
−1
at pH 7.0 and 1.7 μmol of NADH formed min
−1
mg
−1
at pH 9.5. In the presence of the protonophore carbonyl cyanide
m-
chlorophenylhydrazone and the Na
+
/H
+
antiporter monensin, the H
2
-dependent reduction of NAD
+
by membrane vesicles decreased only slightly (about 16%). The NADP
+
- or NAD
+
-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-
N
-oxide or Triton X-100. NAD(P)H formation with H
2
as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by
K. pneumoniae
during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H
2
:NAD(P)
+
oxidoreductase that provides reducing equivalents for the synthesis of cell material.
Repair of Oxidized Bases in the Extremely Radiation-Resistant Bacterium
Deinococcus radiodurans
Authors: Bauche Cécile , Laval Jacques
Published: December 2019

Deinococcus radiodurans
is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that
D. radiodurans
possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase–a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the
Escherichia coli
Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.
APT1
, but Not
APT2
, Codes for a Functional Adenine Phosphoribosyltransferase in
Saccharomyces cerevisiae
Authors: Alfonzo Juan D. , Crother Timothy R. , Guetsova Maria L. , Daignan-Fornier Bertrand , Taylor Milton W.
Published: December 2019

The yeast
Saccharomyces cerevisiae
has two separate genes (
APT1
and
APT2
) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only
APT1
could code for a complementing activity. Cloning and expression of both the
APT1
and
APT2
genes in
Escherichia coli
showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only
APT1
was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that
APT1
is the functional gene in
S. cerevisiae
and that
APT2
is a pseudogene.
A Novel
Bacillus subtilis
Gene,
antE
, Temporally Regulated and Convergent to and Overlapping
dnaE
Authors: Wang Lin-Fa , Park Sung-Soo , Doi Roy H.
Published: December 2019

A
Bacillus subtilis
promoter, Px, that functions in a convergent manner with the
sigA
operon promoter P3 has been found in the
sigA
operon. Promoter Px is turned on at the same time as promoter P3 during early sporulation. The transcript from promoter Px codes for a small protein with partial homology to the OmpR protein from
Escherichia coli
and also carries an untranslated sequence at its 3′ end that is complementary to the 5′ end of the P3 transcript, which codes for the ribosome binding site of
dnaE
. The gene controlled by Px has been called
antE
. The expression of
antE
does not require ς
B
, ς
E
, or ς
H
. Px was transcribed in vitro by the ς
A
holoenzyme and is the seventh promoter to be recognized in the ς
A
operon. A possible role for the
antE
gene during early sporulation is proposed.
Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA
Authors: Ueda Kumiko , Seki Tatsuji , Kudo Takuji , Yoshida Toshiomi , Kataoka Masakazu
Published: December 2019

To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475
Streptomyces
strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity.
Growth of
Azospirillum irakense
KBC1 on the Aryl β-Glucoside Salicin Requires either
salA
or
salB
Authors: Faure Denis , Desair Jos , Keijers Veerle , Bekri My Ali , Proost Paul , Henrissat Bernard , Vanderleyden Jos
Published: December 2019

The rhizosphere nitrogen-fixing bacterium
Azospirillum irakense
KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes,
salA
and
salB
, conferring β-glucosidase activity to
Escherichia coli
, have been identified in a cosmid library of
A. irakense
DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(
salA-salB
)
A. irakense
mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either
salA
or
salB
, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.
A HilA-Independent Pathway to
Salmonella typhimurium
Invasion Gene Transcription
Authors: Rakeman Jennifer L. , Bonifield Heather R. , Miller Samuel I.
Published: December 2019

Salmonella typhimurium
invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within
Salmonella
pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci,
sirB
and
sirC
, which contribute to SPI1 gene transcription were defined.
sirC
is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype.
sirB
is required for maximal expression of
sirC
and consists of two open reading frames located near
kdsA
, a gene involved in lipopolysaccharide biosynthesis.
sirC
expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of
sirC
or
sirA
restores expression of a subset of SPI1 genes, including
invF
and
sspC
, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of
inv
expression is important for efficient assembly and function of the SPI1 TTSS.
Bacteriophage T4
rnh
(RNase H) Null Mutations: Effects on Spontaneous Mutation and Epistatic Interaction with
rII
Mutations
Authors: Bebenek Anna , Smith Leslie A. , Drake John W.
Published: December 2019

The bacteriophage T4
rnh
gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4
rnh
null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Ac
r
) mutations. Because mutations in the related
Saccharomyces cerevisiae RAD27
gene display a remarkable duplication mutator phenotype, we further explored the impact of
rnh
mutations upon the mutation process. We observed that most Ac
r
mutants in an
rnh
+
strain contain
ac
mutations, whereas only roughly half of the Ac
r
mutants detected in an
rnh
Δ strain bear
ac
mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of
ac
mutations arising in
rnh
Δ and
rnh
+
backgrounds are indistinguishable. Thus, the increase in Ac
r
mutants in an
rnh
Δ background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of
rI
mutations in an
rnh
Δ background. In a screen that detects mutations at both the
rI
locus and the much larger
rII
locus, the r frequency was severalfold lower in an
rnh
Δ background. This decrease was due to the phenotype of
rnh rII
double mutants, which display an r
+
plaque morphology but retain the characteristic inability of
rII
mutants to grow on λ lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process.
Genetic Diversity of the Streptococcal Competence (
com
) Gene Locus
Authors: Whatmore Adrian M. , Barcus Victoria A. , Dowson Christopher G.
Published: December 2019

The
com
operon of naturally transformable streptococcal species contains three genes,
comC
,
comD
, and
comE
, involved in the regulation of competence. The
comC
gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The
comD
and
comE
genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the
comD
-encoded protein being a receptor for CSP. Here we report on the genetic variability of
comC
and
comD
within
Streptococcus pneumoniae
isolates. Comparative analysis of sequence variations of
comC
and
comD
shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many
S. pneumoniae
strains in the laboratory, there is a clear correlation between the presence of a particular
comC
allele and the cognate
comD
allele. These findings effectively rule out the possibility that the presence of noncognate
comC
and
comD
alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional
com
pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of
S. mitis
and
S. oralis
. The CSPs from these isolates are much more closely related to those from
S. pneumoniae
than to most CSPs previously reported from
S. mitis
and
S. oralis
, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of
S. pneumoniae
that are under strong selective pressure.
Cell Cycle Expression and Transcriptional Regulation of DNA Topoisomerase IV Genes in
Caulobacter
Authors: Ward Doyle V. , Newton Austin
Published: December 2019

DNA replication and differentiation are closely coupled during the
Caulobacter crescentus
cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the
parE
and
parC
genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897–910, 1997). We have examined the cell cycle regulation of
parE
and
parC
and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of
parE
and
parC
expression is not strictly coordinated, however. The rate of
parE
transcription increases ca. 20-fold during the G
1
-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the
parC
promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through
parE
expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major
Caulobacter
sigma factor ς
73
. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the −10 and −35 elements of the
parE
and
parC
promoters is required for maximum levels of
parE
transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of
parE
transcription and the
parE
and
parC
promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication.
Saccharomyces cerevisiae
Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of the
PKC1-MPK1
Cell Integrity Pathway
Authors: Ketela Troy , Green Robin , Bussey Howard
Published: December 2019

The
MID2
gene of
Saccharomyces cerevisiae
encodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor.
MID2
was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of
MID2
causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression of
MID2
causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. α-Factor hypersensitivity of
mid2
Δ mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, including
PKC1
,
RHO1
,
WSC1
, and
WSC2
. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since
mid2Δ wsc1
Δ mutants are inviable on medium that does not contain osmotic support. A role for
MID2
in the cell integrity pathway is further supported by the finding that
MID2
is required for induction of Mpk1p tyrosine phosphorylation during exposure to α-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame,
MTL1
, which encodes a protein with both structural and functional similarity to Mid2p.
Redundant In Vivo Proteolytic Activities of
Escherichia coli
Lon and the ClpYQ (HslUV) Protease
Authors: Wu Whi-Fin , Zhou YanNing , Gottesman Susan
Published: December 2019

The ClpYQ (HslUV) ATP-dependent protease of
Escherichia coli
consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA;
lon
mutants are UV sensitive, due to the stabilization of SulA.
lon
mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of
clpY
or
clpQ
leads to further stabilization of SulA in a
lon
mutant but not in
lon
+
cells. While either
lon
,
lon clpY
, or
lon clpQ
mutants are UV sensitive at low temperatures, at elevated temperatures the
lon
mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions.
Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA from
Escherichia coli
Authors: Koebnik Ralf
Published: December 2019

The N-terminal domain of the OmpA protein from
Escherichia coli
, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel β-barrel whose eight transmembrane β-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral β-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest β-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.
Proliferation of Intrahyphal Hyphae Caused by Disruption ofcsmA, Which Encodes a Class V Chitin Synthase with a Myosin Motor-Like Domain inAspergillus nidulans
Authors: Horiuchi Hiroyuki , Fujiwara Makoto , Yamashita Shuichi , Ohta Akinori , Takagi Masamichi
Published: December 2019

We have found that theAspergillus nidulans csmAgene encodes a novel protein which consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain (M. Fujiwara, H. Horiuchi, A. Ohta, and M. Takagi, Biochem. Biophys. Res. Commun. 236:75–78, 1997). To clarify the roles ofcsmAin fungal morphogenesis, we constructedcsmAnull mutants. The growth rate of the mutant colonies was almost the same as that of the wild-type strain, but hyphal growth was severely inhibited when a chitin-binding reagent, Calcofluor white or Congo red, was added to the medium. Moreover, morphological abnormalities in tip growth and septum formation were identified microscopically. Proliferation of intracellular new hyphae, called intrahyphal hyphae, which behaved as intrinsic hyphae, was the most striking phenotypic feature among them. These phenotypes were not suppressed when the only chitin synthase domain ofcsmAwas expressed under the control of thealcApromoter, whereas they were suppressed when the intact form ofcsmAwas expressed. Therefore, it was concluded that the product ofcsmA(CsmA) has important roles in polarized cell wall synthesis and maintenance of cell wall integrity and that the myosin motor-like domain is indispensable for these functions.
A Mutant
Escherichia coli
Primase Defective in Elongation of Primer RNA Chains
Authors: Sun Wuliang , Schoneich Jerzy , Godson G. Nigel
Published: December 2019

Earlier we showed by affinity cross-linking of initiating substrates to
Escherichia coli
primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A. A. Mustaev and G. N. Godson, J. Biol. Chem. 270:15711–15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5′ CTG 3′ pRNA initiation site on phage G4 oric DNA. The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.
Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal PathogenPorphyromonas gingivalis
Authors: DeCarlo Arthur A. , Paramaesvaran Mayuri , Yun Peter L. W. , Collyer Charles , Hunter Neil
Published: December 2019

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogenPorphyromonas gingivalis. We therefore examined the potential role of the dominantP. gingivaliscysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd= 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki= 2.5 μM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd= 2.1 nM) was also inhibited by protoporphyrin IX (Ki= 10 μM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released byP. gingivaliscells in a batch culture, in parallel with proteinase activity. Cysteine proteinases fromP. gingivalisappear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition inP. gingivalismay allow precise targeting of this critical metabolic aspect for periodontal disease prevention.
An Intracellular Iron Chelator Pleiotropically Suppresses Enzymatic and Growth Defects of Superoxide Dismutase-Deficient
Escherichia coli
Authors: Maringanti Sujatha , Imlay James A.
Published: December 2019

Mutants of
Escherichia coli
that lack cytoplasmic superoxide dismutase (SOD) exhibit auxotrophies for sulfur-containing, branched-chain, and aromatic amino acids and cannot catabolize nonfermentable carbon sources. A secondary-site mutation substantially relieved all of these growth defects. The requirement for fermentable carbon and the branched-chain auxotrophy occur because superoxide (O
2
−
) leaches iron from the [4Fe-4S] clusters of a family of dehydratases, thereby inactivating them; the suppression of these phenotypes was mediated by the restoration of activity to these dehydratases, evidently without changing the intracellular concentration of O
2
−
. Cloning, complementation, and sequence analysis identified the suppressor mutation to be in
dapD
, which encodes tetrahydrodipicolinate succinylase, an enzyme involved in diaminopimelate and lysine biosynthesis. A block in
dapB
, which encodes dihydrodipicolinate reductase in the same pathway, conferred similar protection. Genetic analysis indicated that the protection stems from the intracellular accumulation of tetrahydro- or dihydrodipicolinate. Heterologous expression in the SOD mutants of the dipicolinate synthase of
Bacillus subtilis
generated dipicolinate and similarly protected them. Dipicolinates are excellent iron chelators, and their accumulation in the cell triggered derepression of the Fur regulon and a large increase in the intracellular pool of free iron, presumably as a dipicolinate chelate. A
fur
mutation only partially relieved the auxotrophies, indicating that Fur derepression assists but is not sufficient for suppression. It seems plausible that the abundant internal iron permits efficient reactivation of superoxide-damaged iron-sulfur clusters. This result provides circumstantial evidence that the sulfur and aromatic auxotrophies of SOD mutants are also directly or indirectly linked to iron metabolism.
Construction and Initial Characterization of
Escherichia coli
Strains with Few or No Intact Chromosomal rRNA Operons
Authors: Asai Tsuneaki , Condon Ciarán , Voulgaris Justina , Zaporojets Dmitry , Shen Binghua , Al-Omar Michaal , Squires Craig , Squires Catherine L.
Published: December 2019

The
Escherichia coli
genome carries seven rRNA (
rrn
) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these
rrn
deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the
rrn
deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.
Identification of Two New Proteins in Spermidine Nucleoids Isolated from
Escherichia coli
Authors: Murphy Lizabeth D. , Rosner Judah L. , Zimmerman Steven B. , Esposito Dominic
Published: December 2019

The
Escherichia coli
nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type
E. coli
. Partial amino-terminal sequencing has identified them as the products of
rdgC
and
yejK
. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.
Fused and Overlapping
rpoB
and
rpoC
Genes in Helicobacters, Campylobacters, and Related Bacteria
Authors: Zakharova Natalya , Paster Bruce J. , Wesley Irene , Dewhirst Floyd E. , Berg Douglas E. , Severinov Konstantin V.
Published: December 2019

The genes coding for the β (
rpoB
) and β′ (
rpoC
) subunits of RNA polymerase are fused in the gastric pathogen
Helicobacter pylori
but separate in other taxonomic groups. To better understand how the unique fused structure evolved, we determined DNA sequences at and around the
rpoB-rpoC
junction in 10 gastric and nongastric species of
Helicobacter
and in members of the related genera
Wolinella
,
Arcobacter
,
Sulfurospirillum
, and
Campylobacter
. We found the fusion to be specific to
Helicobacter
and
Wolinella
genera;
rpoB
and
rpoC
overlap in the other genera. The fusion may have arisen by a frameshift mutation at the site of
rpoB
and
rpoC
overlap. Loss of good Shine-Dalgarno sequences might then have fixed the fusion in the
Helicobacteraceae
, even if fusion itself did not confer a selective advantage.
Effects of Ethyl and Benzyl Analogues of Spermine on
Escherichia coli
Peptidyltransferase Activity, Polyamine Transport, and Cellular Growth
Authors: Karahalios Panagiotis , Amarantos Ioannis , Mamos Petros , Papaioannou Dionysios , Kalpaxis Dimitrios L.
Published: December 2019

Various ethyl and benzyl spermine analogues, including the anticancer agent
N
1
,
N
12
-bis(ethyl)spermine, were studied for their ability to affect the growth of cultured
Escherichia coli
cells, to inhibit [
3
H]putrescine and [
3
H]spermine uptake into cells, and to modulate the peptidyltransferase activity (EC 2. 3. 2. 12). Relative to other cell lines, growth of
E. coli
was uniquely insensitive to these analogues. Nevertheless, these analogues conferred similar modulation of in vitro protein synthesis and inhibition of [
3
H]putrescine and [
3
H]spermine uptake, as is seen in other cell types. Thus, both ethyl and benzyl analogues of spermine not only promote the formation and stabilization of the initiator ribosomal ternary complex, but they also have a sparing effect on the Mg
2+
requirements. Also, in a complete cell-free protein-synthesizing system, these analogues at low concentrations stimulated peptide bond formation, whereas at higher concentrations, they inhibited the reaction. The ranking order for stimulation of peptide-bond formation by the analogues was
N
4
,
N
9
-dibenzylspermine >
N
4
,
N
9
-bis(ethyl)spermine ≅
N
1
-ethylspermine >
N
1
,
N
12
-bis(ethyl)spermine, whereas the order of analogue potency regarding the inhibitory effect was inverted, with inhibition constant values of 10, 3.1, 1.5, and 0.98 μM, respectively. Although the above analogues failed to interact with the putrescine-specific uptake system, they exhibited high affinity for the polyamine uptake system encoded by the
potABCD
operon. Despite this fact, none of the analogues could be internalized by the polyamine transport system, and therefore they could not influence the intracellular polyamine pools and growth of
E. coli
cells.
Structure-Function Analysis of XcpP, a Component Involved in General Secretory Pathway-Dependent Protein Secretion inPseudomonas aeruginosa
Authors: Bleves Sophie , Gérard-Vincent Manon , Lazdunski Andrée , Filloux Alain
Published: December 2019

The general secretory pathway ofPseudomonas aeruginosais required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. AnxcpPmutant demonstrated that the product of this gene is indeed an essential element of theP. aeruginosasecretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.
Energy Conservation by the H2:Heterodisulfide Oxidoreductase fromMethanosarcina mazeiGö1: Identification of Two Proton-Translocating Segments
Authors: Ide Tina , Bäumer Sebastian , Deppenmeier Uwe
Published: December 2019

The membrane-bound H2:heterodisulfide oxidoreductase system of the methanogenic archaeonMethanosarcina mazeiGö1 catalyzed the H2-dependent reduction of 2-hydroxyphenazine and the dihydro-2-hydroxyphenazine-dependent reduction of the heterodisulfide of HS-CoM and HS-CoB (CoM-S-S-CoB). Washed inverted vesicles of this organism were found to couple both processes with the transfer of protons across the cytoplasmic membrane. The maximal H+/2e−ratio was 0.9 for each reaction. The electrochemical proton gradient (ΔμH+) thereby generated was shown to drive ATP synthesis from ADP plus Pi, exhibiting stoichiometries of 0.25 ATP synthesized per two electrons transported for both partial reactions. ATP synthesis and the generation of ΔμH+were abolished by the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847). The ATP synthase inhibitorN,N′-dicyclohexylcarbodiimide did not affect H+translocation but led to an almost complete inhibition of ATP synthesis and decreased the electron transport rates. The latter effect was relieved by the addition of SF 6847. Thus, the energy-conserving systems showed a stringent coupling which resembles the phenomenon of respiratory control. The results indicate that two different proton-translocating segments are present in the H2:heterodisulfide oxidoreductase system; the first involves the 2-hydroxyphenazine-dependent hydrogenase, and the second involves the heterodisulfide reductase.
Molecular Cloning and Functional Expression of Alternative Oxidase from
Candida albicans
Authors: Huh Won-Ki , Kang Sa-Ouk
Published: December 2019

The
AOX1
gene, which encodes an alternative oxidase, was isolated from the genomic DNA library of
Candida albicans
. The gene encodes a polypeptide consisting of 379 amino acids with a calculated molecular mass of 43,975 Da. The
aox1/aox1
mutant strain did not show cyanide-resistant respiration under normal conditions but could still induce cyanide-resistant respiration when treated with antimycin A. The measurement of respiratory activity and Western blot analysis suggested the presence of another AOX. When
C. albicans AOX1
was expressed in alternative oxidase-deficient
Saccharomyces cerevisiae
, it could confer cyanide-resistant respiration on
S. cerevisiae
.
Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-Dependent Glycerol Dehydratase
Authors: Tobimatsu Takamasa , Kajiura Hideki , Yunoki Michio , Azuma Muneaki , Toraya Tetsuo
Published: December 2019

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of
Klebsiella pneumoniae
, the inactivated enzyme is reactivated in the presence of ATP, Mg
2+
, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them
gdrA
and
gdrB
. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant
Escherichia coli
cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.
Mutations in Catabolite Control Protein CcpA Separating Growth Effects from Catabolite Repression
Authors: Küster Elke , Hilbich Tanja , Dahl Michael K. , Hillen Wolfgang
Published: December 2019

Carbon catabolite repression in
Bacillus megaterium
is mediated by the transcriptional regulator CcpA. A chromosomal deletion of
ccpA
eliminates catabolite repression and reduces the growth rate on glucose. We describe four single-amino-acid mutations in CcpA which separate the growth effect from catabolite repression, suggesting distinct regulatory pathways for these phenotypes.
Isolation and Characterization of Mutations in the
Escherichia coli
Regulatory Protein XapR
Authors: Jørgensen Casper , Dandanell Gert
Published: December 2019

In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in
Escherichia coli
. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from
Klebsiella aerogenes
, are placed close together and are most probably directly involved in binding the inducer xanthosine.
Identification and Complementation of Frameshift Mutations Associated with Loss of Cytadherence in
Mycoplasma pneumoniae
Authors: Fisseha Makda , Göhlmann Hinrich W. H. , Herrmann Richard , Krause Duncan C.
Published: December 2019

Mycoplasma pneumoniae
cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of
hmw2
by Tn
4001
results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in
hmw2
. In this study, the recombinant wild-type
hmw2
allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I
hmw2
allele did not restore cytadherence, consistent with a defect in
hmw2
in this mutant. A frameshift was discovered in different oligoadenine tracts in
hmw2
from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in
hmw2
. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.
Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of
Staphylococcus aureus
Bacteriophage 187
Authors: Loessner Martin J. , Gaeng Susanne , Scherer Siegfried
Published: December 2019

We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique
Staphylococcus aureus
phage 187. The endolysin gene
ply187
encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined
ply187
domains. This enzymatically active N terminus showed convincing amino acid sequence homology to an
N
-acetylmuramoyl-
l
-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of
ply187
and was shown to encode a small, hydrophobic protein of holin-like function. The
hol187
gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in
Escherichia coli
resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected
S. aureus
cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ
S
am mutation in a nonsuppressing
E. coli
HB101 background. Plaque formation by λgt11::
hol187
indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of
ply187
and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected
Staphylococcus
cells.