bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Replacement Analysis of the
Streptomyces virginiae barA
Gene Encoding the Butyrolactone Autoregulator Receptor Reveals that BarA Acts as a Repressor in Virginiamycin Biosynthesis
Authors: Nakano Hiroko , Takehara Emio , Nihira Takuya , Yamada Yasuhiro
Published: December 2019

Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of
Streptomyces
species, act as a primary signal in
Streptomyces virginiae
to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two
barA
mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp
Eco
T14I fragment of
barA
was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type
S. virginiae
on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in
S. virginiae
. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain NH2, an internal
Eco
NI-
Sma
I fragment of
barA
was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutated
barA
genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.
Expression of the
bglH
Gene of
Lactobacillus plantarum
Is Controlled by Carbon Catabolite Repression
Authors: Marasco Rosangela , Muscariello Lidia , Varcamonti Mario , De Felice Maurilio , Sacco Margherita
Published: December 2019

A newly identified
bglH
gene coding for a phospho-β-glucosidase of
Lactobacillus plantarum
was isolated and expressed in
Escherichia coli
. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The
bglH
gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in
L. plantarum
cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of
bglH
expression.
The A
1
A
o
ATPase from
Methanosarcina mazei
: Cloning of the 5′ End of the
aha
Operon Encoding the Membrane Domain and Expression of the Proteolipid in a Membrane-Bound Form in
Escherichia coli
Authors: Ruppert Claudia , Wimmers Sönke , Lemker Thorsten , Müller Volker
Published: December 2019

Three additional ATPase genes, clustered in the order
ahaH
,
ahaI
, and
ahaK
, were found upstream of the previously characterized genes
ahaECFABDG
coding for the archaeal A
1
A
o
ATPase from
Methanosarcina mazei
.
ahaH
, the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis revealed that the clusters
ahaHIK
and
ahaECFABDG
are transcribed as one message. AhaH is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in
Methanobacterium thermoautotrophicum
and
Methanococcus jannaschii
. AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane α helices. It is similar to the 100-kDa polypeptide of V
1
V
o
ATPases and is therefore suggested to participate in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane α helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from
M. thermoautotrophicum
and
M. jannaschii
, respectively.
ahaK
is expressed in
Escherichia coli
, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria. This is the first report on the expression and incorporation into
E. coli
lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase.
Does Disparate Occurrence of Autoregulatory Programmed Frameshifting in Decoding the Release Factor 2 Gene Reflect an Ancient Origin with Loss in Independent Lineages?
Authors: Persson Britt C. , Atkins John F.
Published: December 2019

In
Escherichia coli
an autoregulatory mechanism of programmed ribosomal frameshifting governs the level of polypeptide chain release factor 2. From an analysis of 20 sequences of genes encoding release factor 2, we infer that this frameshift mechanism was present in a common ancestor of a large group of bacteria and has subsequently been lost in three independent lineages.
The Glycyl Radical Enzyme TdcE Can Replace Pyruvate Formate-Lyase in Glucose Fermentation
Authors: Sawers Gary , Heßlinger Christian , Muller Nathalie , Kaiser Manuela
Published: December 2019

Mutants of
Escherichia coli
unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the
pfl
gene from
Clostridium butyricum
, we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an
E. coli pfl
mutation. The clostridial gene encoded a basic 14.5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn
554
from
Staphylococcus aureus
. Our studies revealed that the clostridial TcbC protein activated the transcription of the
E. coli tdcABCDEFG
operon, which encodes an anaerobic
l
-threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when
E. coli
grows in the absence of catabolite-repressing sugars and in the presence of
l
-threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the
tdc
operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Heßlinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477–492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to
pfl
mutants.
Isolation and Characterization of
Bacillus subtilis sigB
Operon Mutations That Suppress the Loss of the Negative Regulator RsbX
Authors: Smirnova Natalya , Scott Janelle , Voelker Uwe , Haldenwang W. G.
Published: December 2019

ς
B
, a transcription factor that controls the
Bacillus subtilis
general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress. RsbX, one of seven ς
B
regulators (Rsb proteins) whose genes are cotranscribed with ς
B
, is a negative regulator in the stress-dependent activation pathway. To better define the interactions that take place among the Rsb proteins, we analyzed
sigB
operon mutations which suppress the high-level ς
B
activity that normally accompanies the loss of RsbX. Each of these mutations was in one of three genes (
rsbT
, -
U
, and -
V
) which encode positive regulators of ς
B
, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate. ς
B
activity remained inducible by ethanol in several of the RsbX
−
suppressor strains. This finding supports the notion that RsbX is not needed as the target for ς
B
activation by at least some stresses. ς
B
activity in several RsbX
−
strains with suppressor mutations in
rsbT
or -
U
was high during growth and underwent a continued, rather than a transient, increase following stress. Thus, RsbX is likely responsible for maintaining low ς
B
activity during balanced growth and for reestablishing ς
B
activity at prestress levels following induction. Although RsbX likely participates in limiting the ς
B
induction response, a second mechanism for curtailing unrestricted ς
B
activation was suggested by the ς
B
induction profile in two suppressor strains with mutations in
rsbV
. ς
B
activity in these mutants was stress inducible but transient, even in the absence of RsbX.
Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis
Authors: Abouhamad Walid N. , Bray Dennis , Schuster Martin , Boesch Kristin C. , Silversmith Ruth E. , Bourret Robert B.
Published: December 2019

Escherichia coli
responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except
cheA
and
cheY
resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the
che
genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.
Species-Dependent Phenotypes of Replication-Temperature-Sensitive
trfA
Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of
trfA
Expression
Authors: Karunakaran Ponniah , Blatny Janet Martha , Ertesvåg Helga , Valla Svein
Published: December 2019

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four
trfA
mutants temperature sensitive for replication in
Pseudomonas aeruginosa
. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in
Escherichia coli
. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in
P. aeruginosa
, and the effect of the neutral mutation was caused by its ability to strongly reduce the
trfA
expression level. The double mutant and mutant 163C could not be stably maintained in
E. coli
, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the
trfA
expression level also in
E. coli
. The glycine 85 codon in
trfA
mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type
trfA
was expressed at much lower levels in
E. coli
than in
P. aeruginosa
, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The
E. coli lacZ
gene was translationally fused at the 3′ end and internally in
trfA
, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.
Identification of an Intragenic Ribosome Binding Site That Affects Expression of the
uncB
Gene of the
Escherichia coli
Proton-Translocating ATPase (
unc
) Operon
Authors: Matten Sharlene R. , Schneider Thomas D. , Ringquist Steven , Brusilow William S. A.
Published: December 2019

The
uncB
gene codes for the a subunit of the F
o
proton channel sector of the
Escherichia coli
F
1
F
o
ATPase. Control of expression of
uncB
appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997–3011, 1982) identified a potential ribosome binding site within the
uncB
reading frame preceding a five-codon reading frame which is shifted one base relative to the
uncB
reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an
uncB
′-′
lacZ
fusion gene containing most of
uncB
. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of
lacZ
to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of
uncB
is reduced by translational frameshifting and/or a translational false start at this site within the
uncB
reading frame.
Genetic Analysis of Dioxin Dioxygenase of
Sphingomonas
sp. Strain RW1: Catabolic Genes Dispersed on the Genome
Authors: Armengaud Jean , Happe Birgitta , Timmis Kenneth N.
Published: December 2019

The dioxin dioxygenase of
Sphingomonas
sp. strain RW1 activates dibenzo-
p
-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The
dxnA1
and
dxnA2
cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of
dxnA1A2
is modulated according to the available carbon source, expression of the
dbfB
gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of
dxnA1A2
but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of
Sphingomonas
sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the α and β subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in
Escherichia coli
cells containing the cloned
dxnA1A2
gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-
p
-dioxin was obtained. The dioxin dioxygenase was not active in
E. coli
cells coexpressing a class IIB electron supply system. In the course of the isolation of the
dxnA1
and
dxnA2
cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genus
Sphingomonas
, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as
Pseudomonas
species.
A Dual-Signaling Mechanism Mediated by the ArcB Hybrid Sensor Kinase Containing the Histidine-Containing Phosphotransfer Domain in
Escherichia coli
Authors: Matsushika Akinori , Mizuno Takeshi
Published: December 2019

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows
Escherichia coli
to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the
sdh
operon. The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of
sdh
. Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype with respect to ArcB-ArcA signaling. The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of
sdh
. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.
Multicellular Stalk-Like Structures in
Saccharomyces cerevisiae
Authors: Engelberg David , Mimran Avishai , Martinetto Horacio , Otto Joel , Simchen Giora , Karin Michael , Fink Gerald R.
Published: December 2019

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (
Candida albicans
,
Schizosaccharomyces pombe
, and
Escherichia coli
) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.
Role of PhoP∼P in Transcriptional Regulation of Genes Involved in Cell Wall Anionic Polymer Biosynthesis in
Bacillus subtilis
Authors: Qi Ying , Hulett F. Marion
Published: December 2019

tagA
,
tagD
, and
tuaA
operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in
Bacillus subtilis
. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation of
tuaA
and the repression of
tagA
and
tagD
. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the
tagA
and
tagD
promoters and also to activate the transcription of the
tuaA
promoter.
Molecular Characterization of a Chromosomal Determinant Conferring Resistance to Zinc and Cobalt Ions in
Staphylococcus aureus
Authors: Xiong Anming , Jayaswal Radheshyam K.
Published: December 2019

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of
Staphylococcus aureus
RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated
zntR
and
zntA
. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of
zntA
was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing
zntR
and
zntA
. An
S. aureus
strain harboring multiple copies of
zntR
and
zntA
showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that
zntR
and
zntA
genes were cotranscribed. The zinc uptake studies suggested that the
zntA
product was involved in the export of zinc ions out of cells.
A Redox-Responsive Pathway for Aerobic Regulation of Photosynthesis Gene Expression in
Rhodobacter sphaeroides
2.4.1
Authors: O’Gara James P. , Eraso Jesus M. , Kaplan Samuel
Published: December 2019

To further understand the proposed signal transduction pathway involving the presumed redox proteins RdxBH and
cbb
3
cytochrome oxidase in
Rhodobacter sphaeroides
2.4.1, a series of mutants lacking components of both the Prr two-component activation system and the
cbb
3
-type cytochrome oxidase or RdxBH were constructed. We report that under highly aerobic conditions, aberrant photosynthesis gene expression and spectral complex formation typical of
cbb
3
- or RdxBH-deficient mutants were no longer observed when either
prrA
(encoding the response regulator of the Prr system) or
prrB
(encoding the presumed sensor kinase) was also deleted. These double-mutant strains are phenotypically identical to single-mutant PrrA and PrrB strains, suggesting that the signal(s) originating from the
cbb
3
terminal oxidase affects downstream
puc
and
puf
operon expression by acting exclusively through the Prr system. When the same double-mutant strains were examined under anaerobic dark dimethyl sulfoxide growth conditions, photosynthesis gene expression was obligatorily linked to the two-component activation system. However, photosynthesis gene expression under the same growth conditions was significantly higher in the
cbb
3
mutant strain when compared to that in the wild type. Similarly, under anaerobic photosynthetic conditions the high levels of the oxidized carotenoid, spheroidenone, which accumulate in
cbb
3
-deficient mutants were nearly restored to normal in a PrrB
−
CcoP
−
double mutant. This observation, together with previously published results, suggests that the regulation of the CrtA-catalyzed reaction possesses both transcriptional and posttranscriptional regulatory effectors. We propose that the
cbb
3
cytochrome oxidase, which by definition can interact with external oxygen, serves to control the activity of the Prr two-component activation system under both aerobic and anaerobic conditions. Although independent from the
cbb
3
oxidase, the RdxBH proteins are also required for normal functioning of the Prr two-component activation system and are therefore believed to lie between the
cbb
3
oxidase in this oxygen-sensing, redox signaling pathway and the Prr activation system.
Branched-Chain Amino Acid Biosynthesis in
Salmonella typhimurium
: a Quantitative Analysis
Authors: Epelbaum Sabine , LaRossa Robert A. , VanDyk Tina K. , Elkayam T. , Chipman David M. , Barak Ze’ev
Published: December 2019

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in
Salmonella typhimurium
LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.
Structural and Functional Analysis of the Gene Cluster Encoding the Enzymes of the Arginine Deiminase Pathway of
Lactobacillus sake
Authors: Zúñiga Manuel , Champomier-Verges Marie , Zagorec Monique , Pérez-Martínez Gaspar
Published: December 2019

Lactobacillus sake
can use arginine via the arginine deiminase (ADI) pathway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the
L. sake
counterpart sequences by PCR. Screening a genomic library of
L. sake
in λEMBL3 allowed us to isolate a clone containing a 10-kb
L. sake
genomic DNA insert. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI (
arcA
), OTC (
arcB
), carbamate kinase (
arcC
), and a putative carrier with high similarity to the arginine/ornithine antiporter of
Pseudomonas aeruginosa
(
arcD
). Additionally, a putative transaminase-encoding gene (
arcT
) was located in this region. The genes followed the order
arcA arcB arcC arcT arcD
, which differs from that found in other microorganisms.
arcA
,
arcB
,
arcC
, and
arcD
mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studies indicated that
arcA
gene is subject to catabolite repression, and under the conditions used, several transcripts could be detected, suggesting the existence of different initiation sites or processing of a larger mRNA.
Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from
Escherichia coli
Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine
Authors: Tourneux Lise , Bucurenci Nadia , Lascu Ioan , Sakamoto Hiroshi , Briand Gilbert , Gilles Anne-Marie
Published: December 2019

The wild-type TMP kinases from
Escherichia coli
and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type
tmk
gene from
Escherichia coli
or
Bacillus subtilis
, but not the defective
tmk
gene, restored the resistance to bromodeoxyuridine of an
E. coli
mutant strain.
Identification and Expression of the
Bacillus subtilis
Fructose-1,6-Bisphosphatase Gene (
fbp
)
Authors: Fujita Yasutaro , Yoshida Ken-Ichi , Miwa Yasuhiko , Yanai Nobuo , Nagakawa Eishi , Kasahara Yasuhiro
Published: December 2019

The
Bacillus subtilis fbp
gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as
yydE
. The
fbp
gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis.
fbp
transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly,
B. subtilis
FBPase exhibited no significant similarity to other FBPases in protein sequence databases.
Efficiency and Frequency of Translational Coupling between the Bacteriophage T4 Clamp Loader Genes
Authors: Torgov Michael Y. , Janzen Deanna M. , Reddy Michael K.
Published: December 2019

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the “sliding clamp” of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the “clamp loader” activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes
44
and
62
, which are directly adjacent on polycistronic mRNA molecules, are—to some degree—translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene
44
. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ∼85% and a coupling frequency of ∼25% between the
44-62
gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.
Chitinolytic Activity in
Chromobacterium violaceum
: Substrate Analysis and Regulation by Quorum Sensing
Authors: Chernin Leonid S. , Winson Michael K. , Thompson Jacquelyn M. , Haran Shoshan , Bycroft Barrie W. , Chet Ilan , Williams Paul , Stewart Gordon S. A. B.
Published: December 2019

Quorum sensing control mediated by
N
-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In
Chromobacterium violaceum
ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL
N
-hexanoyl-
l
-homoserine lactone (HHL). In this study we show that
C. violaceum
produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using
p
-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of
N
-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of
N
-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two
N
-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn
5
mutant of
C. violaceum
(CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the
C. violaceum
wild-type strain or 10 μM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.
Similarities between the
antABC
-Encoded Anthranilate Dioxygenase and the
benABC
-Encoded Benzoate Dioxygenase of
Acinetobacter
sp. Strain ADP1
Authors: Bundy Becky M. , Campbell Alan L. , Neidle Ellen L.
Published: December 2019

Acinetobacter
sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the β-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed the
antABC
genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of
antABC
in
Escherichia coli
, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In
Acinetobacter
mutants,
benC
substituted for
antC
during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the
benAB
genes did not substitute for
antAB
. An
antA
point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a
mucK
mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of
antA
, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity of
antABC
did not prevent the use of anthranilate as a sole carbon source. The
antABC
genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.
Molecular Characterization and Sequence of a Methionine Biosynthetic Locus from
Pseudomonas syringae
Authors: Andersen Gary L. , Beattie Gwyn A. , Lindow Steven E.
Published: December 2019

Two methionine biosynthetic genes in
Pseudomonas syringae
pv.
syringae
,
metX
and
metW
, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The
metXW
locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine
O
-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus
Leptospira
, and several gram-positive bacterial genera. Both
metX
and
metW
were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including
metX
and
metW
mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.
Vibrio cholerae
O139 Bengal: Combined Physical and Genetic Map and Comparative Analysis with the Genome of
V. cholerae
O1
Authors: Khetawat Gopal , Bhadra Rupak K. , Kar Sujata , Das Jyotirmoy
Published: December 2019

A combined physical and genetic map of the genome of strain SG24 of
Vibrio cholerae
O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes
Not
I,
Sfi
I, and
Ceu
I. The genome of SG24 is circular, and the genome size is about 3.57 Mb. The linkages between 47
Not
I and 32
Sfi
I fragments of
V. cholerae
SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine
Ceu
I fragments were determined primarily by analyses of partial fragments of the
Ceu
I-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a
V. cholerae
genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.
Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase
Authors: Horiuchi Takayuki , Komano Teruya
Published: December 2019

The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the
pilS
product, and a minor component, one of the seven
pilV
products. The
pilS
product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the
pilU
product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the
pilV
product. To reveal the relationship between the structure and function of the
pilS
product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant
pilS
products were analyzed. Four
pilS
mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type
pilS
gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the
pilS
product requires the entire prepilin sequence.
Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in
Burkholderia cepacia
AC1100
Authors: Zaborina Olga , Daubaras Dayna L. , Zago Anna , Xun Luying , Saido Katsuhiko , Klem Thomas , Nikolic Dejan , Chakrabarty A. M.
Published: December 2019

Burkholderia cepacia
AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.
Intracellular α-Amylase of
Streptococcus mutans
Authors: Simpson Christine L. , Russell Roy R. B.
Published: December 2019

Sequencing upstream of the
Streptococcus mutans
gene for a CcpA gene homolog,
regM
, revealed an open reading frame, named
amy
, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of
Streptococcus bovis
and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in
S. mutans
cell extracts, with no activity detected in culture supernatants. Inactivation of
amy
by insertion of an antibiotic resistance marker confirmed that
S. mutans
has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch.
S. mutans
can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of
amy
did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from
S. mutans
, but the
amy
mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the
amy
mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in
S. mutans
grown on maltose.
Mutations in
Salmonella
Pathogenicity Island 2 (SPI2) Genes Affecting Transcription of SPI1 Genes and Resistance to Antimicrobial Agents
Authors: Deiwick Jörg , Nikolaus Thomas , Shea Jaqueline E. , Gleeson Colin , Holden David W. , Hensel Michael
Published: December 2019

The
Salmonella typhimurium
genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of
S. typhimurium
to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of
sipC
,
prgK
, and
hilA
, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.
Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from
Escherichia coli
: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes
Authors: Pompeo Frédérique , van Heijenoort Jean , Mengin-Lecreulx Dominique
Published: December 2019

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from
Escherichia coli
was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The

K
m

values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.
The
Bacteroides fragilis
BtgA Mobilization Protein Binds to the
oriT
Region of pBFTM10
Authors: Sitailo Leonid A. , Zagariya Alexander M. , Arnold Patrick J. , Vedantam Gayatri , Hecht David W.
Published: December 2019

The
Bacteroides fragilis
conjugal plasmid pBFTM10 contains two genes,
btgA
and
btgB
, and a putative
oriT
region necessary for transfer in
Bacteroides fragilis
and
Escherichia coli
. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of
btgA
revealed three sets of inverted repeats, potentially locating the
oriT
region. A 304-bp DNA fragment comprising this putative
oriT
region was cloned and confirmed to be the functional pBFTM10
oriT
by bacterial conjugation experiments using
E. coli
and
B. fragilis. btgA
was cloned and overexpressed in
E. coli
, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate
oriT
. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the
oriT
-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.