bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from
Bacillus thuringiensis
Authors: Wilcks Andrea , Smidt Lasse , Økstad Ole Andreas , Kolstø Anne-Brit , Mahillon Jacques , Andrup Lars
Published: December 2019

A 5.8-kb fragment of the large conjugative plasmid pAW63 from
Bacillus thuringiensis
subsp.
kurstaki
HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63
ori
. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the
Enterococcus faecalis
plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes
repC
and
prgO
, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in
Bacillus subtilis
and
B. thuringiensis
and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other
B. thuringiensis
subsp.
kurstaki
strains and to a strain of
B. thuringiensis
subsp.
alesti
.
In Vitro Selection of Integration Host Factor Binding Sites
Authors: Goodman Steven D. , Velten Nerissa J. , Gao Qian , Robinson Scott , Segall Anca M.
Published: December 2019

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3′ domain is conserved, the 5′ domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5′ domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5′ binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5′ binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.
Characterization of MarR Superrepressor Mutants
Authors: Alekshun Michael N. , Levy Stuart B.
Published: December 2019

MarR negatively regulates expression of the multiple antibiotic resistance (
mar
) locus in
Escherichia coli
. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to
marO
in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.
Isolation and Characterization of Mutations in
Bacillus subtilis
That Allow Spore Germination in the Novel Germinant
d
-Alanine
Authors: Paidhungat Madan , Setlow Peter
Published: December 2019

Bacillus subtilis
spores break their metabolic dormancy through a process called germination. Spore germination is triggered by specific molecules called germinants, which are thought to act by binding to and stimulating spore receptors. Three homologous operons,
gerA
,
gerB
, and
gerK
, were previously proposed to encode germinant receptors because inactivating mutations in those genes confer a germinant-specific defect in germination. To more definitely identify genes that encode germinant receptors, we isolated mutants whose spores germinated in the novel germinant
d
-alanine, because such mutants would likely contain gain-of-function mutations in genes that encoded preexisting germinant receptors. Three independent mutants were isolated, and in each case the mutant phenotype was shown to result from a single dominant mutation in the
gerB
operon. Two of the mutations altered the
gerBA
gene, whereas the third affected the
gerBB
gene. These results suggest that
gerBA
and
gerBB
encode components of the germinant receptor. Furthermore, genetic interactions between the wild-type
gerB
and the mutant
gerBA
and
gerBB
alleles suggested that the germinant receptor might be a complex containing GerBA, GerBB, and probably other proteins. Thus, we propose that the
gerB
operon encodes at least two components of a multicomponent germinant receptor.
Maltose Metabolism in the Hyperthermophilic Archaeon
Thermococcus litoralis
: Purification and Characterization of Key Enzymes
Authors: Xavier Karina B. , Peist Ralf , Kossmann Marina , Boos Winfried , Santos Helena
Published: December 2019

Maltose metabolism was investigated in the hyperthermophilic archaeon
Thermococcus litoralis
. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in
T. litoralis
cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism
Pyrococcus furiosus
used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a
p
-nitrophenyl-α-
d
-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in
T. litoralis
, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in
T. litoralis
were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in
Escherichia coli
and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A

K
m

of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon
Pyrococcus horikoshii
OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium
Thermotoga maritima
(60%) and
Mycobacterium tuberculosis
(31%) but not with that of the enzyme from
E. coli
(13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the
T. litoralis
enzyme.
Growth Phase-Regulated Induction of
Salmonella
-Induced Macrophage Apoptosis Correlates with Transient Expression of SPI-1 Genes
Authors: Lundberg Urban , Vinatzer Ursula , Berdnik Daniela , von Gabain Alexander , Baccarini Manuela
Published: December 2019

Invasive
Salmonella
has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the
Salmonella
pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of
Salmonella
to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the
hilA
gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ.
hilA
itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however,
hilA
was found to be required only for the expression of the
prgH
gene, while
sipB
,
invA
, and
invF
were expressed in a
hilA
-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.
Regulation of Alginate Biosynthesis in
Pseudomonas syringae
pv. syringae
Authors: Fakhr Mohamed K. , Peñaloza-Vázquez Alejandro , Chakrabarty Ananda M. , Bender Carol L.
Published: December 2019

Both
Pseudomonas aeruginosa
and the phytopathogen
P. syringae
produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in
P. syringae
are different from those in
P. aeruginosa
with copper being a major signal in
P. syringae
. In
P. aeruginosa
, the alternate sigma factor encoded by
algT
(ς
22
) and the response regulator AlgR1 are required for transcription of
algD
, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from
P. syringae
. The deduced amino acid sequence of AlgR1 from
P. syringae
showed 86% identity to its
P. aeruginosa
counterpart. Sequence analysis of the region flanking
algR1
in
P. syringae
revealed the presence of
argH
,
algZ
, and
hemC
in an arrangement virtually identical to that reported in
P. aeruginosa
. An
algR1
mutant,
P. syringae
FF5.32, was defective in alginate production but could be complemented when
algR1
was expressed in
trans
. The
algD
promoter region in
P. syringae
(
PsalgD
) was also characterized and shown to diverge significantly from the
algD
promoter in
P. aeruginosa
. Unlike
P. aeruginosa
,
algR1
was not required for the transcription of
algD
in
P. syringae
, and
PsalgD
lacked the consensus sequence recognized by AlgR1. However, both the
algD
and
algR1
upstream regions in
P. syringae
contained the consensus sequence recognized by ς
22
, suggesting that
algT
is required for transcription of both genes.
Regulation of
hmp
Gene Transcription in
Mycobacterium tuberculosis
: Effects of Oxygen Limitation and Nitrosative and Oxidative Stress
Authors: Hu Yanmin , Butcher Philip D. , Mangan Joseph A. , Rajandream Marie-Adele , Coates Anthony R. M.
Published: December 2019

The
Mycobacterium tuberculosis hmp
gene encodes a protein which is homologous to flavohemoglobin in
Escherichia coli
. Northern blotting analysis demonstrated that
hmp
transcription increased when a microaerophilic culture became oxygen limited as it entered stationary phase at 20 days. There was a fivefold increase of the
hmp
transcripts during early stationary phase compared with the value which was observed in the exponential growth phase. This induction of
hmp
transcription was not due to changes in the mRNA stability since the half-life of
hmp
mRNA was very short in a 20-day microaerophilic culture. No induction of
hmp
mRNA was observed during entry into stationary phase when the culture was continuously aerated.
hmp
transcription was induced after a short exposure of a late-exponential-phase culture to anaerobic conditions. These data indicate that oxygen limitation is the trigger for
hmp
gene transcription. In addition, when a microaerophilic culture entered into the stationary phase at 20 days, transcription of
hmp
increased to a small extent after exposure to
S
-nitrosoglutathione (a nitric oxide [NO] releaser) and sodium nitroprusside (an NO
+
donor) and decreased after exposure to paraquat (a superoxide generator) and H
2
O
2
. In log phase (4 days) and late stationary phase (40 days), the transcription of
hmp
was unaffected by nitrosative and oxidative stress. Three primer extension products were observed. The −10 region is 100% identical to that of promoter T3 in mycobacteria and shows a strong similarity to the −10 sequence of
hmp
and
rpoS
promoters in
E. coli
. These observations of
hmp
mRNA induction in response to O
2
limitation and nitrosative stress suggest that the
hmp
gene of
M. tuberculosis
may have a role in protection of the organism from NO killing under microaerophilic conditions.
Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by
Escherichia coli
Authors: London Robert E. , Allen Devon L. , Gabel Scott A. , DeRose Eugene F.
Published: December 2019

We have evaluated the use of [1,2-
13
C
2
]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted
Escherichia coli
MG1655 cells were performed. Preservation of the
13
C-
13
C-
12
C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in
E. coli
. Additionally, formation of
13
C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-
13
C
2
]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-
13
C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2-
13
C
2
]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.
The Glucuronic Acid Utilization Gene Cluster from
Bacillus stearothermophilus
T-6
Authors: Shulami Smadar , Gat Orit , Sonenshein Abraham L. , Shoham Yuval
Published: December 2019

A λ-EMBL3 genomic library of
Bacillus stearothermophilus
T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-
d
-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (
orf1
, -
2
, -
3
, and -
4
) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-
O
-α-(4-
O
-methyl-α-
d
-glucuronosyl)-xylotriose] (MeGlcUAXyl
3
). The following two genes code for an intracellular α-glucuronidase (
aguA
) and a β-xylosidase (
xynB
). Five more genes (
kdgK
,
kdgA
,
uxaC
,
uxuA
, and
uxuB
) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene,
uxuR
, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl
3
, the most likely molecular inducer.
The Morphological Transition ofHelicobacter pyloriCells from Spiral to Coccoid Is Preceded by a Substantial Modification of the Cell Wall
Authors: Costa Katyssulla , Bacher Gerold , Allmaier Günter , Dominguez-Bello María Gloria , Engstrand Lars , Falk Per , de Pedro Miguel A. , García-del Portillo Francisco
Published: December 2019

The peptidoglycan (murein) ofHelicobacter pylorihas been investigated by high-performance liquid chromatography and mass spectrometric techniques. Murein fromH. pyloricorresponded to the A1γ chemotype, but the muropeptide elution patterns were substantially different from the one forEscherichia coliin that the former produced high proportions of muropeptides with a pentapeptide side chain (about 60 mol%), with Gly residues as the C-terminal amino acid (5 to 10 mol%), and with (1→6)anhydro-N-acetylmuramic acid (13 to 18 mol%).H. pylorimurein also lacks murein-bound lipoprotein, trimeric muropeptides, and (l-d) cross-linked muropeptides. Cessation of growth and transition to coccoid shape triggered an increase inN-acetylglucosaminyl-N-acetylmuramyl–l-Ala–d-Glu (approximately 20 mol%), apparently at the expense of monomeric muropeptides with tri- and tetrapeptide side chains. Muropeptides with (1→6)anhydro-muramic acid and with Gly were also more abundant in resting cells.
Transcriptional Organization and In Vivo Role of the
Escherichia coli rsd
Gene, Encoding the Regulator of RNA Polymerase Sigma D
Authors: Jishage Miki , Ishihama Akira
Published: December 2019

The regulator of sigma D (Rsd) was identified as an RNA polymerase ς
70
-associated protein in stationary-phase
Escherichia coli
with the inhibitory activity of ς
70
-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of
rsd
mRNA indicated the presence of two promoters, ς
S
-dependent P1 and ς
70
-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the ς
70
- or ς
S
-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the
rsd
null mutant, the ς
70
- and ς
S
-dependent gene expression was increased or decreased, respectively. On the other hand, the ς
70
- or ς
S
-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the ς
S
-dependent transcription in the
rsd
mutant is overcome by increased production of the ς
S
subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase ς subunit from ς
70
to ς
S
during the transition from exponential growth to the stationary phase.
Transcriptional Regulation in the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Coordinated Expression of Divergently Oriented Genes in Response to β-Linked Glucose Polymers
Authors: Voorhorst Wilfried G. B. , Gueguen Yannick , Geerling Ans C. M. , Schut Gerti , Dahlke Isabell , Thomm Michael , van der Oost John , de Vos Willem M.
Published: December 2019

The genetic organization, expression, and regulation of the
celB
locus of the hyperthermophilic archaeon
Pyrococcus furiosus
were analyzed. This locus includes the
celB
gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster,
adhA-adhB-lamA
, which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the
lamA
operon). During growth of
P. furiosus
on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the
celB
gene and the
lamA
operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the
celB
gene and the
lamA
operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the
celB-adhA
intergenic region, some of which might be part of a transcriptional regulator-binding site.
Analysis of Quorum-Sensing-Dependent Control of Rhizosphere-Expressed (
rhi
) Genes in
Rhizobium leguminosarum
bv. viciae
Authors: Rodelas Belen , Lithgow James K. , Wisniewski-Dye Florence , Hardman Andrea , Wilkinson Adam , Economou Anastassios , Williams Paul , Downie J. Allan
Published: December 2019

The
rhi
genes of
Rhizobium leguminosarum
biovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372–376, 1996) the
rhiABC
operon had been shown to be regulated by RhiR and to be induced by added
N
-(3-hydroxy-7-
cis
-tetradecenoyl)-
l
-homoserine lactone (3OH,C
14:1
-HSL). Mutagenesis of a cosmid carrying the
rhiABC
and
rhiR
gene region identified a gene (
rhiI
) that affects the level of
rhiA
expression. Mutation of
rhiI
slightly increased the number of nodules formed on the pea. The
rhiI
gene is (like
rhiA
) regulated by
rhiR
in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of
N
-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces
N
-(hexanoyl)-
l
-homoserine lactone (C
6
-HSL) and
N
-(octanoyl)-
l
-homoserine lactone (C
8
-HSL). Both of these AHLs induced
rhiA-lacZ
and
rhiI-lacZ
expression on plasmids introduced into an
Agrobacterium
strain that produces no AHLs, showing that
rhiI
is positively regulated by autoinduction. However, in this system no induction of
rhiA
or
rhiI
with 3OH,C
14:1
-HSL was observed. Analysis of the spent culture supernatant of the wild-type
R. leguminosarum
bv. viciae revealed that at least seven different AHLs are made. Mutation of
rhiI
decreased the amounts of C
6
-HSL and C
8
-HSL but did not block their formation, and in this background the
rhiI
mutation did not significantly affect the expression levels of the
rhiI
gene or
rhiABC
genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in
R. leguminosarum
bv. viciae and that they affect
rhiI
and
rhiABC
expression. We postulate that the previously observed induction of
rhiA
by 3OH,C
14:1
-HSL may be due to an indirect effect caused by induction of other AHL production loci.
AGT1
, Encoding an α-Glucoside Transporter Involved in Uptake and Intracellular Accumulation of Trehalose in
Saccharomyces cerevisiae
Authors: Plourde-Owobi Lucile , Durner Sophie , Parrou Jean-Luc , Wieczorke Roman , Goma Gerard , François Jean
Published: December 2019

The trehalose content in
Saccharomyces cerevisiae
can be significantly manipulated by including trehalose at an appropriate level in the growth medium. Its uptake is largely dependent on the expression of
AGT1
, which encodes an α-glucoside transporter. The trehalose found in a
tps1
mutant of trehalose synthase may therefore largely reflect its uptake from the enriched medium that was employed.
Genotype, Phenotype, and Protein Structure in a Regulator of Sporulation: Effects of Mutations in the
spoIIAA
Gene of
Bacillus subtilis
Authors: Barillà Daniela , Lucet Isabelle , Kuhlmann Anne , Yudkin Michael D.
Published: December 2019

SpoIIAA, a phosphorylatable protein, is essential to the regulation of ς
F
, the first sporulation-specific transcription factor of
Bacillus subtilis
. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding
B. subtilis
mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.
Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins
Authors: Hamada Kenji , Terashima Hiromichi , Arisawa Mikio , Yabuki Nami , Kitada Kunio
Published: December 2019

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in
Saccharomyces cerevisiae
is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.
ς
K
Can Negatively Regulate
sigE
Expression by Two Different Mechanisms during Sporulation of
Bacillus subtilis
Authors: Zhang Bin , Struffi Paolo , Kroos Lee
Published: December 2019

Temporal and spatial gene regulation during
Bacillus subtilis
sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade. In the mother cell compartment of sporulating cells, expression of the
sigE
gene, encoding the earlier-acting sigma factor, ς
E
, is negatively regulated by the later-acting sigma factor, ς
K
. Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of
sigE
transcription. Production of ς
K
about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of
sigE
transcription. A mutation in the
spo0A
gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early ς
K
production on
sigE
expression early in sporulation. Also, early production of ς
K
reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent
ald
gene. In contrast, both
sigE
and
ald
were overexpressed late in development of cells that fail to make ς
K
. The
ald
promoter, like the
sigE
promoter, is believed to be recognized by ς
A
RNA polymerase, suggesting that ς
K
may inhibit ς
A
activity late in sporulation. To exert this negative effect, ς
K
must be transcriptionally active. A mutant form of ς
K
that associates with core RNA polymerase, but does not direct transcription of a ς
K
-dependent gene, failed to negatively regulate expression of
sigE
or
ald
late in development. On the other hand, the negative effect of early ς
K
production on
sigE
expression early in sporulation did not require transcriptional activity of ς
K
RNA polymerase. These results demonstrate that ς
K
can negatively regulate
sigE
expression by two different mechanisms, one observed when ς
K
is produced earlier than normal, which does not require ς
K
to be transcriptionally active and affects Spo0A, and the other observed when ς
K
is produced at the normal time, which requires ς
K
RNA polymerase transcriptional activity. The latter mechanism facilitates the switch from ς
E
to ς
K
in the cascade controlling mother cell gene expression.
Isolation and Properties of
Enterococcus hirae
Mutants Defective in the Potassium/Proton Antiport System
Authors: Kakinuma Yoshimi , Igarashi Kazuei
Published: December 2019

A K
+
/H
+
antiporter regulates cytoplasmic pH in
Enterococcus hirae
growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K
+
/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified.
Overexpression ofMethanococcus voltaeFlagellin Subunits inEscherichia coliandPseudomonas aeruginosa: a Source of Archaeal Preflagellin
Authors: Bayley Douglas P. , Jarrell Ken F.
Published: December 2019

Methanococcus voltaeis a flagellated member of theArchaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins ofM. voltaeare predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of aM. voltaeflagellin in the bacteriaEscherichia coliandPseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes ofM. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding theM. voltaeFlaB2 mature protein fused to theP. aeruginosapilin (PilA) leader peptide was constructed and transformed into both wild-typeP. aeruginosaand a prepilin peptidase (pilD) mutant ofP. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in thepilDmutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in theP. aeruginosapilin) and expressed in both wild-type andpilDmutantP. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in thepilDmutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in aP. aeruginosaPilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.
Eikenella corrodens
Phase Variation Involves a Posttranslational Event in Pilus Formation
Authors: Villar Maria T. , Helber Jennifer T. , Hood Becky , Schaefer Michael R. , Hirschberg Rona L.
Published: December 2019

The human pathogen
Eikenella corrodens
synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate
E. corrodens
VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated
pilA1
,
pilA2
,
pilB
, and
hagA
. Both
pilA1
and
pilA2
predict a type IV pilin. The protein predicted by
pilB
shares sequence identity with the
Dichelobacter nodosus
FimB fimbrial assembly protein. The protein predicted by
hagA
resembles a hemagglutinin. The region containing these four ORFs was designated the
pilA
locus. DNA hybridization and sequence analysis showed that the
pilA
locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant
pilA1
transcript initiating upstream of
pilA1
and terminating at a predicted hairpin structure between
pilA1
and
pilA2
was detected by several assays, as was a less abundant read-through transcript encompassing
pilA1
,
pilA2
, and
pilB
. Transcription from the
pilA
locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in
E. corrodens
.
Heterologous Expression of Correctly Assembled Methylamine Dehydrogenase in
Rhodobacter sphaeroides
Authors: Graichen M. Elizabeth , Jones Limei H. , Sharma Bethel V. , van Spanning Rob J. M. , Hosler Jonathan P. , Davidson Victor L.
Published: December 2019

The biosynthesis of methylamine dehydrogenase (MADH) from
Paracoccus denitrificans
requires four genes in addition to those that encode the two structural protein subunits,
mauB
and
mauA
. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of
P. denitrificans
,
mauFBEDACJG
, were placed under the regulatory control of the
coxII
promoter of
Rhodobacter sphaeroides
and introduced into
R. sphaeroides
by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native
mau
promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from
P. denitrificans
. These results indicate that
mauM
and
mauN
are not required for MADH or TTQ biosynthesis and that
mauFBEDACJG
are sufficient for TTQ biosynthesis, since
R. sphaeroides
cannot synthesize TTQ. A similar construct introduced into
Escherichia coli
did not produce detectable MADH activity or accumulation of the
mauB
and
mauA
gene products but did lead to synthesizes of amicyanin, the
mauC
gene product. This finding suggests that active recombinant MADH is not expressed in
E. coli
because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of
R. sphaeroides
and the
coxII
promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in
E. coli
. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other
mau
genes.
Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor
Authors: Cho Eun Hee , Nam Chan-Eun , Alcaraz Renato , Gardner Jeffrey F.
Published: December 2019

Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the
attP
region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and
ihfA
mutant cells. P22 integrative recombination is also efficient in
Escherichia coli
in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.
Genetic and Molecular Analysis of
cglB
, a Gene Essential for Single-Cell Gliding in
Myxococcus xanthus
Authors: Rodriguez Ana M. , Spormann Alfred M.
Published: December 2019

Gliding movements of individual isolated
Myxococcus xanthus
cells depend on the genes of the A-motility system (
agl
and
cgl
genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of
cgl
mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the
cglB
wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed Δ
cglB
mutant were fully restored upon complementation with the
cglB
wild-type allele. Experiments with a
cglB
allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate Δ
cglB
cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.
Lysis and Lysis Inhibition in Bacteriophage T4:
rV
Mutations Reside in the Holin
t
Gene
Authors: Dressman Holly Kloos , Drake John W.
Published: December 2019

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37°C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene
t
and upon one or more rapid-lysis (
r
) genes whose inactivation prevents lysis inhibition.
t
encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized
r
genes, only two,
rI
and
rV
, are clearly obligatory for lysis inhibition. We show here that
rV
mutations are alleles of
t
that probably render the t protein unable to respond to the lysis inhibition signal. The
tr
alleles cluster in the 5′ third of
t
and produce a strong r phenotype, whereas conditional-lethal
t
alleles produce the classical t phenotype (inability to lyse) and other
t
alleles produce additional, still poorly understood phenotypes.
tr
mutations are dominant to
t
+
, a result that suggests specific ways to probe T4 holin function.
Water Transport across Yeast Vacuolar and Plasma Membrane-Targeted Secretory Vesicles Occurs by Passive Diffusion
Authors: Coury Larry A. , Hiller Mark , Mathai John C. , Jones Elizabeth W. , Zeidel Mark L. , Brodsky Jeffrey L.
Published: December 2019

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in
Saccharomyces cerevisiae
; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in
S. cerevisiae
, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.
Autogenous Regulation of the
Bacillus anthracis pag
Operon
Authors: Hoffmaster Alex R. , Koehler Theresa M.
Published: December 2019

Protective antigen (PA) is an important component of the edema and lethal toxins produced by
Bacillus anthracis
. PA is essential for binding the toxins to the target cell receptor and for facilitating translocation of the enzymatic toxin components, edema factor and lethal factor, across the target cell membrane. The structural gene for PA,
pagA
(previously known as
pag
), is located on the 182-kb virulence plasmid pXO1 at a locus distinct from the edema factor and lethal factor genes. Here we show that a 300-bp gene located downstream of
pagA
is cotranscribed with
pagA
and represses expression of the operon. We have designated this gene
pagR
(for protective antigen repressor). Two
pagA
mRNA transcripts were detected in cells producing PA: a short, 2.7-kb transcript corresponding to the
pagA
gene, and a longer, 4.2-kb transcript representing a bicistronic message derived from
pagA
and
pagR
. The 3′ end of the short transcript mapped adjacent to an inverted repeat sequence, suggesting that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of
pagR
. A
pagR
mutant exhibited increased steady-state levels of
pagA
mRNA, indicating that
pagR
negatively controls expression of the operon. Autogenous control of the operon may involve
atxA
, a
trans
-acting positive regulator of
pagA
. The steady-state level of
atxA
mRNA was also increased in the
pagR
mutant. The mutant phenotype was complemented by addition of
pagR
in
trans
on a multicopy plasmid.
Outer Membrane Changes in a Toluene-Sensitive Mutant of Toluene-Tolerant
Pseudomonas putida
IH-2000
Authors: Kobayashi Hideki , Takami Hideto , Hirayama Hisako , Kobata Kuniko , Usami Ron , Horikoshi Koki
Published: December 2019

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant
Pseudomonas putida
IH-2000 by transposon mutagenesis with Tn
5
. The gene disrupted by insertion of Tn
5
was identified as
cyoC
, which is one of the subunits of cytochrome
o
. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of
cyoC
caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.
Escherichia coli
Open Reading Frame 696 Is
idi
, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase
Authors: Hahn Frederick M. , Hurlburt Anthony P. , Poulter C. Dale
Published: December 2019

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In
Escherichia coli
, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the
E. coli
chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase,
idi
, is not clustered with other known genes for enzymes in the isoprenoid pathway.
E. coli
FH12 was constructed by disruption of the chromosomal
idi
gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type
idi
gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that
idi
is a nonessential gene. Although the
V
max
of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in

K
m

s. The
E. coli
protein requires Mg
2+
or Mn
2+
for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
areABC
Genes Determine the Catabolism of Aryl Esters in
Acinetobacter
sp. Strain ADP1
Authors: Jones Rheinallt M. , Collier Lauren S. , Neidle Ellen L. , Williams Peter A.
Published: December 2019

Acinetobacter
sp. strain ADP1 is able to grow on a range of esters of aromatic alcohols, converting them to the corresponding aromatic carboxylic acids by the sequential action of three inducible enzymes: an
areA
-encoded esterase, an
areB
-encoded benzyl alcohol dehydrogenase, and an
areC
-encoded benzaldehyde dehydrogenase. The
are
genes, adjacent to each other on the chromosome and transcribed in the order
areCBA
, were located 3.5 kbp upstream of
benK. benK
, encoding a permease implicated in benzoate uptake, is at one end of the
ben-cat
supraoperonic cluster for benzoate catabolism by the β-ketoadipate pathway. Two open reading frames which may encode a transcriptional regulator,
areR
, and a porin,
benP
, separate
benK
from
areC
. Each
are
gene was individually expressed to high specific activity in
Escherichia coli
. The relative activities against different substrates of the cloned enzymes were, within experimental error, identical to that of wild-type
Acinetobacter
sp. strain ADP1 grown on either benzyl acetate, benzyl alcohol, or 4-hydroxybenzyl alcohol as the carbon source. The substrate preferences of all three enzymes were broad, encompassing a range of substituted aromatic compounds and in the case of the AreA esterase, different carboxylic acids. The
areA
,
areB
, and
areC
genes were individually disrupted on the chromosome by insertion of a kanamycin resistance cassette, and the rates at which the resultant strains utilized substrates of the aryl ester catabolic pathway were severely reduced as determined by growth competitions between the mutant and wild-type strains.