bluuebunny/crossref_metadata_2025 huggingface dataset:
Illegitimate Recombination Induced by Overproduction of DnaB Helicase in
Escherichia coli
Authors: Yamashita Teruhito , Hanada Katsuhiro , Iwasaki Mihoko , Yamaguchi Hirotaka , Ikeda Hideo
Published: December 2019

Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in
Escherichia coli
. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.
Ni
2+
Transport and Accumulation in
Rhodospirillum rubrum
Authors: Watt Richard K. , Ludden Paul W.
Published: December 2019

The
cooCTJ
gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni
2+
transport assay was used to monitor uptake and accumulation of
63
Ni
2+
into
R. rubrum
and to observe the effect of mutations in the
cooC
,
cooT
, and
cooJ
genes on
63
Ni
2+
transport and accumulation. Cells grown either in the presence or absence of CO transported Ni
2+
with a

K
m

of 19 ± 4 μM and a
V
max
of 310 ± 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the
cooCTJ
genes, either individually or all three genes simultaneously, transported Ni
2+
the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn
2+
, Mg
2+
, Ca
2+
, and Zn
2+
showed no inhibition of
63
Ni
2+
transport but Co
2+
, Cd
2+
, and Cu
2+
inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°C), by protonophores (carbonyl cyanide
m
-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (
N
,
N
′-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni
2+
transport.
63
Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated
63
Ni
2+
accumulation coincided with the appearance of CODH activity in the culture, suggesting that the
63
Ni
2+
was accumulating in CODH. The
cooC
,
cooT
, and
cooJ
genes are required for the increased
63
Ni
2+
accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated
63
Ni
2+
like cells unexposed to CO.
MgATP Binding and Hydrolysis Determinants of NtrC, a Bacterial Enhancer-Binding Protein
Authors: Rombel Irene , Peters-Wendisch Petra , Mesecar Andrew , Thorgeirsson Thorgeir , Shin Yeon-Kyun , Kustu Sydney
Published: December 2019

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of
Salmonella typhimurium
forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the ς
54
-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent

K
d

of unphosphorylated NtrC for MgATPγS is 100 μM at 25°C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 μM at 37°C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of ς
54
-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21

ras

and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn
2+
binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.
Molecular Characterization of Genetic Loci Required for Secretion of Exoproducts in
Burkholderia pseudomallei
Authors: DeShazer David , Brett Paul J. , Burtnick Mary N. , Woods Donald E.
Published: December 2019

Previous studies have demonstrated that
Burkholderia pseudomallei
secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC.
The Thermophilic Yeast
Hansenula polymorpha
Does Not Require Trehalose Synthesis for Growth at High Temperatures but Does for Normal Acquisition of Thermotolerance
Authors: Reinders Anke , Romano Ivano , Wiemken Andres , De Virgilio Claudio
Published: December 2019

The
TPS1
gene from
Hansenula polymorpha
, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of
TPS1
rendered
H. polymorpha
cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells’ ability to acquire thermotolerance.
Amino Acid Residues Involved in the Functional Integrity of
Escherichia coli
Methionine Aminopeptidase
Authors: Chiu Chen-Hsiang , Lee Chao-Zong , Lin Kung-Shih , Tam Ming F. , Lin Lih-Yuan
Published: December 2019

Amino acid residues in the metal-binding and putative substrate-binding sites of
Escherichia coli
methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.
Transport of d-Xylose in Lactobacillus pentosus, Lactobacillus casei, andLactobacillus plantarum: Evidence for a Mechanism of Facilitated Diffusion via the Phosphoenolpyruvate:Mannose Phosphotransferase System
Authors: Chaillou Stéphane , Pouwels Peter H. , Postma Pieter W.
Published: December 2019

We have identified and characterized thed-xylose transport system ofLactobacillus pentosus. Uptake ofd-xylose was not driven by the proton motive force generated by malolactic fermentation and requiredd-xylose metabolism. The kinetics ofd-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparentKmof 8.5 mM and aVmaxof 23 nmol min−1mg of dry weight−1. In two mutants ofL. pentosusdefective in the phosphoenolpyruvate:mannose phosphotransferase system, growth ond-xylose was absent due to the lack ofd-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of theL. curvatus manBgene encoding the cytoplasmic EIIBMancomponent of the EIIMancomplex. The EIIMancomplex is also involved ind-xylose transport inL. caseiATCC 393 andL. plantarum80. These two species could transport and metabolized-xylose after transformation with plasmids which expressed thed-xylose-catabolizing genes ofL. pentosus,xylAB. L. caseiandL. plantarummutants resistant to 2-deoxy-d-glucose were defective in EIIManactivity and were unable to transportd-xylose when transformed with plasmids containing thexylABgenes. Finally, transport ofd-xylose was found to be the rate-limiting step in the growth ofL. pentosusand ofL. plantarumandL. caseiATCC 393 containing plasmids coding for thed-xylose-catabolic enzymes, since the doubling time of these bacteria ond-xylose was proportional to the level of EIIManactivity.
Transformations in Flagellar Structure ofRhodobacter sphaeroides and Possible Relationship to Changes in Swimming Speed
Authors: Armitage Judith P. , Pitta Thomas P. , Vigeant Margot A.-S. , Packer Helen L. , Ford Roseanne M.
Published: December 2019

Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops. Free-swimming R. sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions. DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations. However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix. The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament. In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form. This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation. This form was always significantly longer than the coiled or helical form from which it was derived. The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix. Examination of the three-dimensional swimming pattern showed that R. sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops. The rate of acceleration out of stops was also variable. The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells. The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed.
High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis
Authors: Hare Janelle M. , McDonough Kathleen A.
Published: December 2019

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. ThehmsHFRS genes required for CR binding (Crb+) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb+ phenotype. We constructed a recA mutant strain of Y. pestisKIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flankedpgm locus. Two Pgm-associated phenotypes (Crb+and pesticin sensitivity [Psts]) were used as markers for the presence of the pgm locus in the RecA+KIM10+ and RecA− YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 × 10−3) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb+ phenotype was still lost at a high frequency (4.5 × 10−5), although the loss of the Pstsphenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 × 10−7). These RecA-independent Crb− mutants were caused by mutations in both thehmsHFRS locus and in a newly identified gene,hmsT. Nonpigmented Yersinia pseudotuberculosisand Escherichia coli strains transformed with bothhmsT and hmsHFRS became Crb+. This study demonstrates that in a laboratory culture, the Crb+phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS andhmsT loci.
Response of
hya
Expression to External pH in
Escherichia coli
Authors: King Paul W. , Przybyla Alan E.
Published: December 2019

The
hya
operon of
Escherichia coli
is composed of the genes which synthesize uptake hydrogenase isoenzyme 1 (Hyd1). Although
hya
expression and Hyd1 synthesis occur only under anaerobic conditions, Hyd1 is not essential for growth. In this study we used a
hya′-′lacZ
fusion to characterize parameters of anaerobic growth that maximize
hya
expression in an attempt to further elucidate Hyd1 function. We found that the expression pattern of
hya
followed a decline of external pH. In buffered media where the pH value was set, the onset of
hya
expression initiated earlier in growth and reached a greater peak level in acidic than in alkaline medium. When cultures expressing
hya
were shifted from acidic to alkaline conditions,
hya
expression was arrested; shifting from alkaline to acidic conditions stimulated
hya
expression. Maximal expression of
hya
under all growth conditions required the sigma factor RpoS and transcriptional regulators AppY and ArcA. In the absence of RpoS or AppY, the response of
hya
expression onset to external pH was evident and maximal
hya
levels remained greater in acidic than in alkaline medium. However, the absence of ArcA led to a diminished response of expression onset to external pH and the loss of elevated expression at an acidic external pH. The fermentation end product formate slightly altered
hya
expression levels but was not required for
hya
to respond to external pH. In contrast to
hya
expression, the onset of
hyb
operon expression, encoding uptake hydrogenase isoenzyme 2, was constitutive with respect to external pH. However, external pH did affect
hyb
expression levels, which, in contrast to
hya
, were maximal in alkaline rather than acidic medium.
Regulation of
mga
Transcription in the Group A Streptococcus: Specific Binding of Mga within Its Own Promoter and Evidence for a Negative Regulator
Authors: McIver Kevin S. , Thurman Alec S. , Scott June R.
Published: December 2019

Transcription of
mga
, encoding the multiple virulence gene regulator of the group A streptococcus, is positively autoregulated. This regulation requires a DNA region (Pmga) that contains both a promoter proximal to
mga
(P2) and a promoter located further upstream (P1). To determine if Mga has a direct role in this process, its ability to bind to specific sequences within Pmga was tested. A purified fusion of Mga to the C-terminal end of maltose-binding protein (MBP-Mga), encoded by
malE-mga
, was shown previously to bind to the promoter regions of Mga-regulated genes, including
scpA
and
emm
. We report here that MBP-Mga can function in vivo to regulate
emm
and
mga
. Electrophoretic mobility shift assays and DNase I footprinting were used to demonstrate specific binding of MBP-Mga to two ca. 59-bp binding sites in Pmga centered around bases −108 and −180 from the major P2 start of transcription. Mga binding sites from Pemm and PscpA were shown to compete for binding at the two Pmga sites, suggesting that the same domain of Mga interacts at all of these promoter targets. Deletion of the distal Pmga binding site (site I) in vivo resulted in loss of Mga-dependent transcription from the P2 start. However, the same lesion resulted in an increase in P1 transcription that was independent of Mga. This suggests the existence of a repressor of
mga
transcription with a binding site overlapping those of Mga.
Catabolism of Branched-Chain α-Keto Acids in
Enterococcus faecalis
: the
bkd
Gene Cluster, Enzymes, and Metabolic Route
Authors: Ward Donald E. , Ross R. Paul , van der Weijden Coen C. , Snoep Jacky L. , Claiborne Al
Published: December 2019

Genes encoding a branched-chain α-keto acid dehydrogenase from
Enterococcus faecalis
10C1, E1α (
bkdA
), E1β (
bkdB
), E2 (
bkdC
), and E3 (
bkdD
), were found to reside in the gene cluster
ptb-buk-bkdDABC
. The predicted products of
ptb
and
buk
exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from
Clostridium acetobutylicum
. Activity and redox properties of the purified recombinant enzyme encoded by
bkdD
indicate that
E. faecalis
has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the
ptb
gene product expressed in
Escherichia coli
was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of α-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that α-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the
ptb
and
buk
gene products with the concomitant phosphorylation of ADP. In contrast, an
E. faecalis bkd
mutant constructed by disruption of the
bkdA
gene did not benefit from having α-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the
bkd
gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain α-keto acids that was previously unidentified in
E. faecalis.
The
Escherichia coli
NadR Regulator Is Endowed with Nicotinamide Mononucleotide Adenylyltransferase Activity
Authors: Raffaelli Nadia , Lorenzi Teresa , Mariani P. Luigi , Emanuelli Monica , Amici Adolfo , Ruggieri Silverio , Magni Giulio
Published: December 2019

The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The
Escherichia coli nadR
gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.
Acetate Utilization in
Lactococcus lactis
Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance
Authors: Hols Pascal , Ramos Ana , Hugenholtz Jeroen , Delcour Jean , de Vos Willem M. , Santos Helena , Kleerebezem Michiel
Published: December 2019

Acetate was shown to improve glucose fermentation in
Lactococcus lactis
deficient in lactate dehydrogenase.
13
C and
1
H nuclear magnetic resonance studies using [2-
13
C]glucose and [2-
13
C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD
+
regeneration in the absence of lactate dehydrogenase.
Identification of a Plasmid-Borne Locus in
Rhizobium etli
KIM5s Involved in Lipopolysaccharide O-Chain Biosynthesis and Nodulation of
Phaseolus vulgaris
Authors: Vinuesa Pablo , Reuhs Bradley L. , Breton Christelle , Werner Dietrich
Published: December 2019

Screening of derivatives of
Rhizobium etli
KIM5s randomly mutagenized with mTn
5
SS
gusA
30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv
−
Fix
−
phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (α-
d
-GalA-1→4[α-
d
-GalA-1→5]-Kdo) and tetrasaccharide (α-
d
-Gal-1→6[α-
d
-GalA-1→4]-α-
d
-Man-1→5Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-
O
-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in
trans
on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host
Phaseolus vulgaris
. The mTn
5
insertion in KIM-G1 was located at the N terminus of a putative α-glycosyltransferase, which most likely had a polar effect on a putative β-glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the
lpsβ2
gene recently reported for
R. etli
CFN42.
Positive Transcriptional Feedback Controls Hydrogenase Expression in
Alcaligenes eutrophus
H16
Authors: Schwartz Edward , Buhrke Thorsten , Gerischer Ulrike , Friedrich Bärbel
Published: December 2019

The protein HoxA is the central regulator of the
Alcaligenes eutrophus
H16
hox
regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene
hoxA
was analyzed. Screening of an 8-kb region upstream of
hoxA
with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5′ of
hoxA
; the others were correlated with the nickel metabolism genes
hypA1
,
hypB1
, and
hypX
. All four activities were independent of HoxA and of the minor transcription factor ς
54
. Translational fusions revealed that
hoxA
is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the
hox
regulon. Quantitative transcript assays indicated elevated levels of
hyp
mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that P
MBH
, which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of
hoxA
under hydrogenase-derepressing conditions. Thus, expression of the
hox
regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.
Acylhomoserine Lactone Synthase Activity of the
Vibrio fischeri
AinS Protein
Authors: Hanzelka Brian L. , Parsek Matthew R. , Val Dale L. , Dunlap Paul V. , Cronan John E. , Greenberg E. P.
Published: December 2019

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a
Vibrio fischeri
enzyme that catalyzes the synthesis of
N
-(3-oxohexanoyl)-
l
-homoserine lactone from an acyl-acyl carrier protein and
S
-adenosylmethionine. Another
V. fischeri
gene,
ainS
, directs the synthesis of
N
-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the
Vibrio harveyi
LuxM protein. The
luxM
gene is required for the synthesis of
N
-(3-hydroxybutyryl)-
l
-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of
N
-octanoylhomoserine lactone from
S
-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.
Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication
Authors: Pato M. L. , Banerjee M.
Published: December 2019

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the
Escherichia coli
chromosome which might be involved in organizing nucleoid structure, fragments of
E. coli
chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.
Acetyl Coenzyme A Synthetase (ADP Forming) from the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Identification, Cloning, Separate Expression of the Encoding Genes,
acdAI
and
acdBI
, in
Escherichia coli
, and In Vitro Reconstitution of the Active Heterotetrameric Enzyme from Its Recombinant Subunits
Authors: Musfeldt Meike , Selig Martina , Schönheit Peter
Published: December 2019

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P
i
→ acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile
Pyrococcus furiosus
. Isoform I is a heterotetramer (α
2
β
2
) with an apparent molecular mass of 145 kDa, composed of two subunits, α and β, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated
acdAI
and
acdBI
, were identified in the genome of
P. furiosus
. The genes were separately overexpressed in
Escherichia coli
, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from
P. furiosus
.
Two Family B DNA Polymerases from
Aeropyrum pernix
, an Aerobic Hyperthermophilic Crenarchaeote
Authors: Cann Isaac K. O. , Ishino Sonoko , Nomura Norimichi , Sako Yoshihiko , Ishino Yoshizumi
Published: December 2019

DNA polymerase activities in fractionated cell extract of
Aeropyrum pernix
, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (
polA
and
polB
) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product of
polA
gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of
polB
gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of
Pyrodictium occultum
. The deduced amino acid sequence of
A. pernix
Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain
Crenarchaeota
has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.
Cloning and Characterization of a Cryptic Haloacid Dehalogenase fromBurkholderia cepaciaMBA4
Authors: Tsang Jimmy S. H. , Sam Laiju
Published: December 2019

Burkholderia cepaciaMBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively inEscherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene,chd1, was isolated on a 1.7-kbPstI fragment. This fragment contains a functional promoter, because expression ofchd1inE. coliis orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence ofchd1did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.
Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in
Rhodobacter sphaeroides
f. sp.
denitrificans
IL106
Authors: Sabaty Monique , Schwintner Carole , Cahors Sandrine , Richaud Pierre , Verméglio Andre
Published: December 2019

We have cloned the
nap
locus encoding the periplasmic nitrate reductase in
Rhodobacter sphaeroides
f. sp.
denitrificans
IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N
2
O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that
R. sphaeroides
f. sp.
denitrificans
IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N
2
O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.
Interactions of the Integrase Protein of the Conjugative Transposon Tn
916
with Its Specific DNA Binding Sites
Authors: Jia Yunhua , Churchward Gordon
Published: December 2019

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn
916
Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.
Identification of a Regulator That Controls Stationary-Phase Expression of Catalase-Peroxidase in
Caulobacter crescentus
Authors: Rava Paul S. , Somma Laura , Steinman Howard M.
Published: December 2019

Expression of the catalase-peroxidase of
Caulobacter crescentus
, a gram-negative member of the α subdivision of the
Proteobacteria
, is 50-fold higher in stationary-phase cultures than in exponential cultures. To identify regulators of the starvation response, Tn
5
insertion mutants were isolated with reduced expression of a
katG::lacZ
fusion on glucose starvation. One insertion interrupted an open reading frame encoding a protein with significant amino acid sequence identity to TipA, a helix-turn-helix transcriptional activator in the response of
Streptomyces lividans
to the peptide antibiotic thiostrepton, and lesser sequence similarity to other helix-turn-helix regulators in the MerR family. The
C. crescentus
orthologue of
tipA
was named
skgA
(stationary-phase regulation of
katG
). Stationary-phase expression of
katG
was reduced by 70% in the
skgA
::Tn
5
mutant, and stationary-phase resistance to hydrogen peroxide decreased by a factor of 10. Like the wild type, the
skgA
mutant exhibited starvation-induced cross-resistance to heat and acid shock, entered into the helical morphology that occurs after 9 to 12 days in stationary phase, and during exponential growth induced
katG
in response to hydrogen peroxide challenge. Expression of
skgA
increased 5- to 10-fold in late exponential phase.
skgA
is the first regulator of a starvation-induced stress response identified in
C. crescentus
. SkgA is not a global regulator of the stationary-phase stress response; its action encompasses the oxidative stress-hydrogen peroxide response but not acid or heat responses. Moreover, SkgA is not an alternative ς factor, like RpoS, which controls multiple aspects of starvation-induced cross-resistance to stress in enteric bacteria. These observations raise the possibility that regulation of stationary-phase gene expression in this member of the α subdivision of the
Proteobacteria
is different from that in
Escherichia coli
and other members of the γ subdivision.
The “Pro” Sequence of the Sporulation-Specific ς Transcription Factor ς
E
Directs It to the Mother Cell Side of the Sporulation Septum
Authors: Ju Jingliang , Haldenwang W. G.
Published: December 2019

ς
E
, a mother cell-specific transcription factor of sporulating
Bacillus subtilis
, is derived from an inactive precursor protein (pro-ς
E
). Activation of ς
E
occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-ς
E
amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-ς
E
and green fluorescent protein (GFP) to visualize the intracellular location of pro-ς
E
by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-ς
E
::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-ς
E
::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-ς
E
::GFP, and presumably pro-ς
E
, to the mother cell is likely to be the reason why ς
E
activity is restricted to that compartment.
Resistance to Bacitracin as Modulated by an
Escherichia coli
Homologue of the Bacitracin ABC Transporter BcrC Subunit from
Bacillus licheniformis
Authors: Harel Yael Meller , Bailone Adriana , Bibi Eitan
Published: December 2019

A small open reading frame from the
Escherichia coli
chromosome,
bcrC
EC
, encodes a homologue to the BcrC subunit of the bacitracin permease from
Bacillus licheniformis
. We show that disruption of the chromosomal
bcrC
EC
gene causes bacitracin sensitivity and, conversely, that BcrC
EC
confers bacitracin resistance when expressed from a multicopy plasmid.
Chromosome Segregation and Cell Division Defects in
recBC sbcBC ruvC
Mutants of
Escherichia coli
Authors: Zahradka Davor , Vlahović Ksenija , Petranović Mirjana , Petranović Drago
Published: December 2019

The RuvC protein is important for DNA recombination and repair in
Escherichia coli
. The present work shows that a
ruvC
null mutation introduced into a
recBC sbcBC
background causes severe defects in chromosome segregation and cell division. Both defects were found to result from abortive recombination initiated by the RecA protein.
Functional Expression of hMYH, a Human Homolog of the
Escherichia coli
MutY Protein
Authors: Slupska Malgorzata M. , Luther Wendy M. , Chiang Ju-Huei , Yang Hanjing , Miller Jeffrey H.
Published: December 2019

We have previously described the
hMYH
cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885–3892, 1996). Here, we report that the enzyme expressed from an
hMYH
cDNA clone in
Escherichia coli
complements the mutator phenotype in a
mutY
mutant and can remove A from an A · 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A · G mismatch in vitro.
recD sbcB sbcD
Mutants Are Deficient in Recombinational Repair of UV Lesions by RecBC
Authors: Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte
Published: December 2019

In
recD sbcB sbcD
mutants, repair of UV-irradiated DNA is strongly RecF dependent, indicating that RecBC is inactive. This finding suggests that exonuclease V, exonuclease I (SbcB), and the SbcCD nuclease play a redundant role in vivo, which is essential for the recombination activity of the RecBC complex during UV repair.
Molecular Characterization ofeutFMutants ofSalmonella typhimuriumLT2 IdentifieseutFLesions as Partial-Loss-of-FunctiontonBAlleles
Authors: Thomas Michael G. , O’Toole George A. , Escalante-Semerena Jorge C.
Published: December 2019

TheeutFlocus ofSalmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization ofeutFmutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining theeutFlocus, Δ903andeutF1115, are partial-loss-of-functiontonBalleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonBgene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carryingeutFalleles. Molecular analysis of the Δ903allele identified a deletion that resulted in the fusion of the 3′ end oftonBwith the 3′ end oftrpA. In-frame translation of thetonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903showed that the new mutation was a deletion of two nucleotides at thetonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine andeutoperon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, theeutFlocus does not exist.
Cell Wall Teichoic Acid Glycosylation in
Listeria monocytogenes
Serotype 4b Requires
gtcA
, a Novel, Serogroup-Specific Gene
Authors: Promadej N. , Fiedler F. , Cossart P. , Dramsi S. , Kathariou S.
Published: December 2019

We have identified a novel gene,
gtcA
, involved in the decoration of cell wall teichoic acid of
Listeria monocytogenes
serotype 4b with galactose and glucose. Insertional inactivation of
gtcA
brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid. Interestingly, the composition of membrane-associated lipoteichoic acid was not affected. Complementation of the mutants with the cloned
gtcA
in
trans
restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within
L. monocytogenes
, sequences homologous to
gtcA
were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b,
gtcA
appears to be the first member of a bicistronic operon which includes a gene with homology to
Bacillus subtilis rpmE
, encoding ribosomal protein L31. In contrast to
gtcA
, the latter gene appears conserved among all screened serotypes of
L. monocytogenes
.
Regulation of Hexuronate Utilization in
Bacillus subtilis
Authors: Mekjian Kathleen R. , Bryan Edward M. , Beall Bernard W. , Moran Charles P.
Published: December 2019

We have identified a locus essential for galacturonate utilization in
Bacillus subtilis
. Genes homologous to
Escherichia coli
and
Erwinia chrysanthemi
glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the
B. subtilis
chromosome. A mutant of
B. subtilis
containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a ς
A
-dependent promoter,
exu
P1, located upstream from the first ORF. The eighth ORF in this cluster (the
exu
locus) encodes a LacI and GalR homolog that negatively regulated expression from
exu
P1. A 26-bp inverted repeat sequence centered 15 bp downstream from the
exu
P1 start point of transcription acted in
cis
to negatively regulate expression from
exu
P1 under noninducing conditions. Expression from the
exu
P1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A ς
E
-dependent promoter,
exu
P2, was localized between the second and third ORFs and was active during sporulation.
Septal Localization of FtsQ, an Essential Cell Division Protein in
Escherichia coli
Authors: Chen Joseph C. , Weiss David S. , Ghigo Jean-Marc , Beckwith Jon
Published: December 2019

Septation in
Escherichia coli
requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The
gfp-ftsQ
gene complements a null mutation in
ftsQ
. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.
The
phn
Genes of
Burkholderia
sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism
Authors: Laurie Andrew D. , Lloyd-Jones Gareth
Published: December 2019

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical
nah
-like (
nah
,
ndo
,
pah
, and
dox
) sequences. Here we report the description of a divergent set of PAH catabolic genes, the
phn
genes, which although isofunctional to the classical
nah
-like genes, show very low homology. This
phn
locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb
Hin
dIII fragment from phenanthrene-degrading
Burkholderia
sp. strain RP007. The
phn
genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in
Escherichia coli
the recombinant
phn
enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein α and β subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the
phn
catabolic genes are two putative regulatory genes,
phnR
and
phnS
. Sequence homology suggests that
phnS
is a LysR-type transcriptional activator and that
phnR
, which is divergently transcribed with respect to
phnSFECDAcAdB
, is a member of the ς
54
-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS.