bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of an A-Factor-Responsive Repressor for
amfR
Essential for Onset of Aerial Mycelium Formation in
Streptomyces griseus
Authors: Ueda Kenji , Hsheh Chia-Wen , Tosaki Tsutomu , Shinkawa Hidenori , Beppu Teruhiko , Horinouchi Sueharu
Published: December 2019

A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone) is essential for the initiation of aerial mycelium formation in
Streptomyces griseus. amfR
is one of the genes which, when cloned on a low-copy-number plasmid, suppresses the aerial mycelium-negative phenotype of an A-factor-deficient mutant of
S. griseus
. Disruption of the chromosomal
amfR
gene resulted in complete abolition of aerial mycelium formation, indicating that
amfR
is essential for the onset of morphogenesis. Cloning and nucleotide sequencing of the region upstream of
amfR
predicted an operon consisting of
orf5
,
orf4
, and
amfR
. Consistent with this idea, Northern blotting and S1 mapping analyses suggested that these three genes were cotranscribed mainly by a promoter (P
ORF5
) in front of
orf5
. Furthermore, P
ORF5
was active only in the presence of A-factor, indicating that it is A-factor dependent. Gel mobility shift assays showed the presence of a protein (AdpB) able to bind P
ORF5
in the cell extract from an A-factor-deficient mutant but not from the wild-type strain. AdpB was purified to homogeneity and found to bind specifically to the region from −72 to −44 bp with respect to the transcriptional start point. Runoff transcriptional analysis of P
ORF5
with purified AdpB and an RNA polymerase complex isolated from vegetative mycelium showed that AdpB repressed the transcription in a concentration-dependent manner. It is thus apparent that AmfR as a switch for aerial mycelium formation and AdpB as a repressor for
amfR
are members in the A-factor regulatory cascade, leading to morphogenesis.
ClpB in a Cyanobacterium: Predicted Structure, Phylogenetic Relationships, and Regulation by Light and Temperature
Authors: Celerin Martina , Gilpin Andrea A. , Schisler Nicholas J. , Ivanov Alexander G. , Miskiewicz Ewa , Krol Marianna , Laudenbach David E.
Published: December 2019

The sequence of a genomic clone encoding a 100-kDa stress protein of
Plectonema boryanum
(
p
-ClpB) was determined. The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation are not unique to
p
-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kb
p-clpB
transcript and 110-kDa cytosolic
p
-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions. Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs. We have used this information, along with an identity alignment, to redefine the Clp subfamilies.
H-NS Regulates DNA Repair in
Shigella
Authors: Palchaudhuri Sunil , Tominna Brandon , Leon Myron A.
Published: December 2019

We report a new role for H-NS in
Shigella
spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in
Shigella
, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus,
Shigella flexneri
M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The
hns
mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of
S. flexneri
M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in
Shigella
the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic
Escherichia coli
strains (enteroinvasive and enterohemorrhagic
E. coli
) show low survival whether incubated at 30 or 40°C postirradiation.
E. coli
K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected from
E. coli
organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of
E. coli
, require experimental verification in pathogenic strains which were not adapted to irradiation.
Nucleotide Sequence and Characterization of
cdrA
, a Cell Division-Related Gene of
Helicobacter pylori
Authors: Takeuchi Hiroaki , Shirai Mutsunori , Akada Junko K. , Tsuda Masataka , Nakazawa Teruko
Published: December 2019

We identified cell division-related gene
cdrA
in
Helicobacter pylori
HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of
H. pylori
26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a
cdrA
-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained
cdrA
mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a
cdrA
-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the
cdrA
-disrupted mutant remained mostly as short rods. Furthermore, the
cdrA
-disrupted mutant regained the filamentation phenotype when the intact
cdrA
gene was introduced by allelic exchange. Taken together, these observations show that the
cdrA
gene plays an important role in the cell growth of
H. pylori
.
Cloning and Characterization of
PRA1
, a Gene Encoding a Novel pH-Regulated Antigen of
Candida albicans
Authors: Sentandreu Maria , Elorza M. Victoria , Sentandreu Rafael , Fonzi William A.
Published: December 2019

Candida albicans
is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of
C. albicans
was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated
PRA1
, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The
PRA1
protein was homologous to surface antigens of
Aspergillus
spp. which react with serum from aspergillosis patients, suggesting that the
PRA1
protein may have a role in the host-parasite interaction during candidal infection.
Expression Cloning of a
Pseudomonas
Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase
Authors: Dotson Garry D. , Kaltashov Igor A. , Cotter Robert J. , Raetz Christian R. H.
Published: December 2019

UDP-
N
-acetylglucosamine-3-
O
-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of the
lpxA
gene of
Pseudomonas aeruginosa
from a library of
Pseudomonas
strain PAO1 expressed in
Escherichia coli
LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991).
Pseudomonas lpxA
encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the
E. coli
transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in
E. coli
. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of
P. aeruginosa
DNA. The
lpxA
gene region was localized to a 1.3-kb
Sal
I-
Pst
I fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new
lpxA
homolog. The predicted
Pseudomonas
LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of
E. coli
LpxA. The
P. aeruginosa
UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the
E. coli
enzyme. A plasmid (pGD3) containing the 1.3-kb
Sal
I-
Pst
I fragment complemented
E. coli
RO138, a temperature-sensitive mutant harboring
lpxA2
. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]
−
1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.
Identification of therrmAGene Encoding the 23S rRNA m1G745 Methyltransferase inEscherichia coliand Characterization of an m1G745-Deficient Mutant
Authors: Gustafsson Claes , Persson Britt C.
Published: December 2019

AnEscherichia colimutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmAgene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on theE. colichromosome and identified to be identical to the previously sequenced geneyebH.
Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the
aroP
Gene of
Escherichia coli
Authors: Wang Peixiang , Yang Ji , Ishihama Akira , Pittard A. J.
Published: December 2019

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the
Escherichia coli aroP
gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for
aroP
expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the
aroP
regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.
Relationship between Spontaneous Aminoglycoside Resistance in
Escherichia coli
and a Decrease in Oligopeptide Binding Protein
Authors: Kashiwagi Keiko , Tsuhako Maria Heloisa , Sakata Kaori , Saisho Tomoko , Igarashi Atsuko , Pinto da Costa Sérgio Olavo , Igarashi Kazuei
Published: December 2019

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of
Escherichia coli
were investigated. Among 20 colonies obtained from 10
8
cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the
oppA
gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the
oppA
gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.
Identification of Chlorobenzene Dioxygenase Sequence Elements Involved in Dechlorination of 1,2,4,5-Tetrachlorobenzene
Authors: Beil Stefan , Mason Jeremy R. , Timmis Kenneth N. , Pieper Dietmar H.
Published: December 2019

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities.
Escherichia coli
cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large α-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific
todC1
α-subunit subsequences by equivalent sequences of the
tecA1
α-subunit. Substrate transformation specificities and rates by
E. coli
resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.
Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of
Lactobacillus plantarum
Authors: Glaasker Erwin , Heuberger Esther H. M. L. , Konings Wil N. , Poolman Bert
Published: December 2019

The accumulation of quaternary ammonium compounds in
Lactobacillus plantarum
is mediated via a single transport system with a high affinity for glycine betaine (apparent

K
m

of 18 μM) and carnitine and a low affinity for proline (apparent

K
m

of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in
L. plantarum
and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by
trans
substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.
An ABC Transporter Plays a Developmental Aggregation Role in
Myxococcus xanthus
Authors: Ward Mandy J. , Mok Kenny C. , Astling David P. , Lew Helen , Zusman David R.
Published: December 2019

Myxococcus xanthus
is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other
M. xanthus
response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the
abcA
gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of
abcA
indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the
abcA
gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of
abcA
showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.
The Highly Conserved, Coregulated
SNO
and
SNZ
Gene Families in
Saccharomyces cerevisiae
Respond to Nutrient Limitation
Authors: Padilla Pamela A. , Fuge Edwina K. , Crawford Matthew E. , Errett Allison , Werner-Washburne Margaret
Published: December 2019

SNZ1
, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three
SNZ
genes in
Saccharomyces cerevisiae
, each of which has another highly conserved gene, named
SNO
(
SNZ
proximal open reading frame), upstream. The DNA sequences and relative positions of
SNZ
and
SNO
genes have been phylogenetically conserved. This report details studies of the expression of the
SNZ-SNO
gene pairs under various conditions and phenotypic analysis of
snz-sno
mutants. An analysis of total RNA was used to determine that adjacent
SNZ-SNO
gene pairs are coregulated.
SNZ2/3
and
SNO2/3
mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when
SNZ1
and
SNO1
are induced. In
snz2 snz3
mutants,
SNZ1
mRNA is induced prior to the diauxic shift, when
SNZ2/3
mRNAs are normally induced. Under nitrogen-limiting conditions,
SNZ1
mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients. Strains carrying deletions in all
SNZ-SNO
gene pairs are viable, but
snz1
and
sno1
mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen. The conservation of sequence and chromosomal position, the coregulation and pattern of expression of
SNZ1
and
SNO1
genes, and the sensitivity of
snz1
and
sno1
mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation.
The SecA Subunit of
Escherichia coli
Preprotein Translocase Is Exposed to the Periplasm
Authors: Eichler Jerry , Wickner William
Published: December 2019

SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.
Topological Analysis of
Chlamydia trachomatis
L2 Outer Membrane Protein 2
Authors: Mygind Per , Christiansen Gunna , Birkelund Svend
Published: December 2019

Using monospecific polyclonal antisera to different parts of
Chlamydia trachomatis
L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when
Chlamydia
elementary bodies are treated with dithiothreitol, and protease digestions indicate that Omp2 has a possible two-domain structure.
Nucleotide Excision Repair in the Third Kingdom
Authors: Ögrünç Müge , Becker Donald F. , Ragsdale Stephen W. , Sancar Aziz
Published: December 2019

Nucleotide excision repair, a general repair mechanism for removing DNA damage, is initiated by dual incisions bracketing the lesion. In procaryotes, the dual incisions result in excision of the damage in 12- to 13-nucleotide-long oligomers, and in eucaryotes they result in excision of the damage in the form of 24- to 32-nucleotide-long oligomers. We wished to find out if
Archaea
perform excision repair. Using cell extracts from
Methanobacterium thermoautotrophicum
, we found that this organism removes UV-induced (6-4) photoproducts in the form of 10- to 11-mers by incising the sixth to seventh phosphodiester bond 5′ to the damage and the fourth phosphodiester bond 3′ to the damage.
Escherichia coli
DNA Topoisomerase I and Suppression of Killing by Tn
5
Transposase Overproduction: Topoisomerase I Modulates Tn
5
Transposition
Authors: Yigit Hesna , Reznikoff William S.
Published: December 2019

Tn
5
transposase (Tnp) overproduction is lethal to
Escherichia coli
. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing,
stkD10
, is localized in
topA
(the gene for topoisomerase I). The
stkD10
mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in
rpoH
,
stkA14
(H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704–1713, 1997), can be explained by an increase in
topA
expression; and third, overexpression of wild-type
topA
partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn
5
transposition up to 30-fold in vivo.
A Combination of Three Mutations,
dcd
,
pyrH
, and
cdd
, Establishes Thymidine (Deoxyuridine) Auxotrophy in
thyA
+
Strains of
Salmonella typhimurium
Authors: Krogan Nevan J. , Zaharik Michelle L. , Neuhard Jan , Kelln Rod A.
Published: December 2019

The
dum
gene of
Salmonella typhimurium
was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305–316, 1977). In the genetic background used in their selection, the joint acquisition of a
dcd
(dCTP deaminase) and a
dum
mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that
dum
is identical to
pyrH
, the gene encoding UMP kinase. The level of UMP kinase activity in the
dum
mutant was found to be only 30% of that observed for the
dum
+
strain. Thymidine prototrophy was restored to the original
dum dcd
mutant (KP1361) either by transduction using a
pyrH
+
donor or by complementation with either of two
pyrH
+
-carrying plasmids. Thymidine auxotrophy could be reconstructed in the
dum
+
derivative (KP1389) by the introduction of a mutant
pyrH
allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in
thyA
+
strains, a
dcd
::Km null mutation was constructed. In the wild-type background,
dcd
::Km alone or in combination with a
pyrH
(
dum
) mutation did not result in a thymidine requirement. A third mutation,
cdd
(cytidine-deoxycytidine deaminase), was required together with the
dcd
and
pyrH
mutations to impart thymidine auxotrophy.
Autolysis ofLactococcus lactisIs Influenced by Proteolysis
Authors: Buist Girbe , Venema Gerard , Kok Jan
Published: December 2019

The autolysin AcmA ofLactococcus lactiswas shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced whenL. lactisMG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis ofL. lactisby AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in anL. lactis acmAdeletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.
Effect of
minCD
on FtsZ Ring Position and Polar Septation in
Bacillus subtilis
Authors: Levin Petra Anne , Shim John Jaeyoung , Grossman Alan D.
Published: December 2019

We examined the pattern of FtsZ localization in a
Bacillus subtilis minCD
mutant. When grown in minimal medium, the majority (∼89%) of the
minCD
mutant cells with an FtsZ ring had a single, medially positioned FtsZ ring. These results indicate that genes in addition to
minCD
function to restrict the number and position of FtsZ rings. When grown in rich medium, greater than 50% of the
minCD
mutant cells had multiple FtsZ rings, indicating significant differences in regulation of FtsZ ring formation based on growth medium.
Sequence Divergence of Seryl-tRNA Synthetases in Archaea
Authors: Kim Hyun-soo , Vothknecht Ute C. , Hedderich Reiner , Celic Ivana , Söll Dieter
Published: December 2019

The genomic sequences of
Methanococcus jannaschii
and
Methanobacterium thermoautotrophicum
contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNA
Cys
is formed by direct aminoacylation or by a transformation of serine misacylated to tRNA
Cys
. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from
M. thermoautotrophicum
; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of
M. thermoautotrophicum
and
M. jannaschii
. In addition, SerRS was expressed from a cloned
Methanococcus maripaludis serS
gene. The two enzymes charged serine to their homologous tRNAs and also accepted
Escherichia coli
tRNA as substrate for aminoacylation. Gel shift experiments showed that
M. thermoautotrophicum
SerRS did not mischarge tRNA
Cys
with serine. This indicates that Cys-tRNA
Cys
is formed by direct acylation in these organisms.
Inhibition of Development of
Myxococcus xanthus
by Eukaryotic Protein Kinase Inhibitors
Authors: Jain Ritu , Inouye Sumiko
Published: December 2019

Myxococcus xanthus
is a social bacterium that lives in the soil and undergoes spectacular development to form multicellular fruiting bodies. It contains a large family of eukaryote-like serine/threonine protein kinases. We found that a number of inhibitors for eukaryotic protein serine, threonine, and tyrosine kinases could inhibit the development and sporulation of
M. xanthus
to various degrees. These results suggest that serine/threonine and tyrosine phosphorylation may be involved in development of
M. xanthus
. None of the inhibitors tested had any effect on vegetative growth of
M. xanthus
. Most of them seemed to act during the early stages of development. However, the expression of a very early development-specific gene, Ω4521, was not significantly affected by the inhibitors. The patterns of protein phosphorylation during development were also not significantly altered by the inhibitors, suggesting that the targets of the inhibitors are minor or unstable phosphoproteins but play key roles in fruiting-body formation in
M. xanthus.
The
yvyD
Gene of
Bacillus subtilis
Is under Dual Control of ς
B
and ς
H
Authors: Drzewiecki Kathrin , Eymann Christine , Mittenhuber Gerhard , Hecker Michael
Published: December 2019

During a search by computer-aided inspection of two-dimensional (2D) protein gels for ς
B
-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the
yvyD
gene of
Bacillus subtilis
. In addition to the typical ς
B
-dependent, stress- and starvation-inducible pattern,
yvyD
is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the
sigB
and/or
spo0H
gene, two promoters were mapped upstream of the
yvyD
gene. The ς
B
-dependent promoter drives expression of
yvyD
under stress conditions and after glucose starvation, whereas a ς
H
-dependent promoter is responsible for
yvyD
transcription following amino acid limitation. Analysis of Northern blots revealed that
yvyD
is transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and
relA
mutant cells showed that the YvyD protein is not synthesized in the
relA
mutant background. It was concluded that the stringent response plays a role in the activation of ς
H
. The
yvyD
gene product is homologous to a protein which might modify the activity of ς
54
in gram-negative bacteria. The expression of a ς
L
-dependent (ς
L
is the equivalent of ς
54
in
B. subtilis
)
levD-lacZ
fusion is upregulated twofold in a
yvyD
mutant. This indicates that the
yvyD
gene product, being a member of both the ς
B
and ς
H
regulons, might negatively regulate the activity of the ς
L
regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the ς
B
and ς
H
regulons on one side and the ς
L
regulon on the other.
Comparison In Vitro of a High- and a Low-Abundance Chemoreceptor of
Escherichia coli
: Similar Kinase Activation but Different Methyl-Accepting Activities
Authors: Barnakov Alexander N. , Barnakova Ludmila A. , Hazelbauer Gerald L.
Published: December 2019

In
Escherichia coli
, high-abundance chemoreceptors are present in cellular amounts approximately 10-fold greater than low-abundance chemoreceptors. Cells containing only low-abundance receptors exhibit abnormally low tumble frequencies and do not migrate effectively in spatial gradients. These defects reflect an inherent activity difference between the two receptor classes. We used in vitro assays to investigate this difference. The low-abundance receptor Trg mediated an ∼100-fold activation of the kinase CheA, only twofold less than activation by the high-abundance receptor Tar. In contrast, Trg was less than 1/20 as active as Tar for in vitro methylation. As observed for high-abundance receptors, kinase activation by Trg varied with the extend of modification at methyl-accepting sites; low methylation corresponded to low kinase activation. Thus, in Trg-only cells, low receptor methylation would result in low kinase activation, correspondingly low content of phospho-CheY, and a decreased dynamic range over which attractant binding could modulate kinase activity. These features could account for the low tumble frequency and inefficient taxis exhibited by Trg-only cells. Thus, the crucial functional difference between the receptor classes is likely to be methyl-accepting activity. We investigated the structural basis for this functional difference by introducing onto the carboxy terminus of Trg a CheR-binding pentapeptide, usually found only at the carboxy termini of high-abundance receptors. This addition enhanced the in vitro methyl-accepting activity of Trg 10-fold.
Identification of
Campylobacter jejuni
Promoter Sequences
Authors: Wösten Marc M. S. M. , Boeve Miranda , Koot Mirjam G. A. , van Nuenen Ad C. , van der Zeijst Bernard A. M.
Published: December 2019

A promoterless
lacZ
shuttle vector, which allowed screening of promoters by β-galactosidase activity in
Campylobacter jejuni
and
Escherichia coli
, was developed. Chromosomal DNA fragments from
C. jejuni
were cloned into this vector; 125 of 1,824 clones displayed promoter activity in
C. jejuni
. Eleven clones with strong promoter activity in
C. jejuni
were further characterized. Their nucleotide sequences were determined, and the transcriptional start sites of the putative promoters in
C. jejuni
were determined by primer extension. Only 6 of these 11 promoters were functional in
E. coli
. The 11 newly characterized and 10 previously characterized
C. jejuni
promoters were used to establish a consensus sequence for
C. jejuni
promoters. The 21 promoters were found to be very similar. They contain three conserved regions, located approximately 10, 16, and 35 bp upstream of the transcriptional start point. The −10 region resembles that of a typical ς
70
E. coli
promoter, but the −35 region is completely different. In addition a −16 region typical for gram-positive bacteria was identified.
The
lytE
Gene of
Bacillus subtilis
168 Encodes a Cell Wall Hydrolase
Authors: Margot Philippe , Wahlen Michael , Gholamhuseinian Ahmad , Piggot Patrick , Karamata Dimitri
Published: December 2019

Bacillus subtilis
cell wall-bound protein CWBP33 is encoded by
lytE
, a gene expressed during the exponential growth phase. Sequence analysis of LytE, a 33-kDa protein, reveals two domains. The N-terminal domain contains a threefold-repeated motif common to several peptidoglycan binding proteins, while the C-terminal domain, probably carrying the catalytic activity, has homology with certain exoproteins. Zymographs unambiguously reveal that the absence of CWBP33, due to inactivation of
lytE
, is accompanied by the loss of a lytic activity. In
lytE
mutants, the cell autolysis rate is significantly decreased, although autolysis of corresponding, purified cell walls does not seem to be affected.
Roles of FtsA and FtsZ in Activation of Division Sites
Authors: Begg Ken , Nikolaichik Yevgeny , Crossland Nicola , Donachie William D.
Published: December 2019

Increasing FtsZ induces the formation of minicells at cell poles but does not increase the frequency or timing of central divisions. A coordinate increase in both FtsZ and FtsA, however, increases the frequency of both polar and central divisions.
Ordered Cloned DNA Map of the Genome of
Vibrio cholerae
569B and Localization of Genetic Markers
Authors: Chatterjee Soma , Mondal Asim K. , Begum Nasim A. , Roychoudhury Susanta , Das Jyotirmoy
Published: December 2019

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of
Vibrio cholerae
has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of
V. cholerae
genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the
V. cholerae
genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by
Not
I,
Sfi
I, and
Ceu
I macrorestriction maps. Twenty-seven cloned genes, 9
rrn
operons, and 10 copies of a repetitive DNA sequence (IS
1004
) have been positioned on the ordered cloned DNA map.
New Flagellin-Specifying Genes in Some
Escherichia coli
Strains
Authors: Ratiner Yuli A.
Published: December 2019

Data for further development of the flagellar antigen genetics of the species
Escherichia coli
are reported. Two new flagellin genes named
fllA
and
flmA
were found in
E. coli
781-55, E2987-73, and E223-69, the test strains for
E. coli
flagellar antigens H44, H55, and H54, respectively (collection of the International
Escherichia
and
Klebsiella
Centre of the World Health Organization, Copenhagen, Denmark). Two alleles of
fllA
were identified that encode flagellar antigens H44 (
fllA
44
) and H55 (
fllA
55
), and the only
flmA
allele found (
flmA
54
) encodes antigen H54. The sites of their integration in the
E. coli
K-12 chromosome after P1-mediated transduction were approximately determined and found to be separate from each other and from the known regions of flagellar genes of
E. coli
and salmonellae. The region of
flm
54
was found to repress the expression of some alleles of the flagellin gene
fliC
. In addition, cryptic genes encoding antigens H4 and H38 were found in phenotypically monophasic test strains 781-55 and E2987-73, respectively.
Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from
Rhodococcus opacus
1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence
Authors: Eulberg Dirk , Kourbatova Elena M. , Golovleva Ludmila A. , Schlömann Michael
Published: December 2019

Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strain
Rhodococcus opacus
(
erythropolis
) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The
clcB
gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of
clcR
but separated from it by 222 bp, we detected the
clcA
and
clcD
genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS
1031
and Tn
4811
. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of
R. opacus
1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.
Growth Phase and Temperature Influence Promoter Activity, Transcript Abundance, and Protein Stability during Biosynthesis of thePseudomonas syringaePhytotoxin Coronatine
Authors: Budde Ina P. , Rohde Bettina H. , Bender Carol L. , Ullrich Matthias S.
Published: December 2019

The plant-pathogenic bacteriumPseudomonas syringaepv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18°C, whereas no detectable toxin is produced at 28°C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression ofcmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for thecmaABToperon during bacterial growth at 18 and 28°C. Promoter activity and transcription rates were maximal when cells were incubated at 18°C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18°C than at 28°C. Western blot analysis indicated that CmaB accumulated in the late stationary phase ofP. syringaecultures grown at 18°C but not in cultures incubated at 28°C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18°C than at 28°C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.
External Loops at the C Terminus of
Erwinia chrysanthemi
Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway
Authors: Lindeberg Magdalen , Boyd Carol M. , Keen Noel T. , Collmer Alan
Published: December 2019

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens
Erwinia chrysanthemi
and
Erwinia carotovora
secrete several isozymes of pectate lyase (Pel) by the
out
-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed
pel
genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of
E. chrysanthemi out
genes carried on cosmid pCPP2006 enables
Escherichia coli
to secrete
E. chrysanthemi
, but not
E. carotovora
, Pels. We exploited the high sequence similarity between
E. chrysanthemi
PelC and
E. carotovora
Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by
E. coli
(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.
Characterization of
Rhizobium leguminosarum
Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif
Authors: Finnie Christine , Zorreguieta Angeles , Hartley Nigel M. , Downie J. Allan
Published: December 2019

The
prsDE
genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from
Rhizobium leguminosarum
bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (
plyA
and
plyB
) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a
prsD
(protein secretion) mutant. The
plyA
gene is upstream of
prsD
but appears to be expressed at a very low level (if at all) in cultured bacteria. A
plyB
::Tn
5
mutant has a very large reduction in degradation of EPS and CMC. Cultures of
plyB
mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a
plyA plyB
double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca
2+
-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.