bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome P460 Genes from the Methanotroph
Methylococcus capsulatus
Bath
Authors: Bergmann David J. , Zahn James A. , Hooper Alan B. , DiSpirito Alan A.
Published: December 2019

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium
Nitrosomonas europaea
(R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium
Methylococcus capsulatus
Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from
M. capsulatus
Bath that contains
cyp
, the gene encoding cytochrome P460.
cyp
is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (
cyp
) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from
M. capsulatus
Bath and
N. europaea
was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal,
c
-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using
cyp
indicated that a cytochrome P460 similar to that from
M. capsulatus
Bath may be present in the type II methanotrophs
Methylosinus trichosporium
OB3b and
Methylocystis parvus
OBBP but not in the type I methanotrophs
Methylobacter marinus
A45,
Methylomicrobium albus
BG8, and
Methylomonas
sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from
M. capsulatus
Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.
Novel Organization of the Genes for Phthalate Degradation from
Burkholderia cepacia
DBO1
Authors: Chang Hung-Kuang , Zylstra Gerben J.
Published: December 2019

Burkholderia cepacia
DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (
ophA1
) is present by itself, while the genes for an inactive transporter (
ophD
) and 4,5-dihydroxyphthalate decarboxylase (
ophC
) are linked and the genes for phthalate dioxygenase oxygenase (
ophA2
) and
cis
-phthalate dihydrodiol dehydrogenase (
ophB
) are linked.
ophA1
and
ophDC
are adjacent to each other but are transcribed in opposite directions, while
ophA2B
is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for
ophD
are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.
The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid
Authors: Moll Gert N. , Konings Wil N. , Driessen Arnold J. M.
Published: December 2019

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-
sn
-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant
k
of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.
Cytochrome
bd
Biosynthesis in
Bacillus subtilis
: Characterization of the
cydABCD
Operon
Authors: Winstedt Lena , Yoshida Ken-Ichi , Fujita Yasutaro , von Wachenfeldt Claes
Published: December 2019

Under aerobic conditions
Bacillus subtilis
utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in
B. subtilis
: a
caa
3
-, an
aa
3
-, and a
bd
-type oxidase. We report here the cloning of the structural genes (
cydA
and
cydB
) encoding the cytochrome
bd
complex. Downstream of the structural genes,
cydC
and
cydD
are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of
cydA
or deletion of
cydABCD
resulted in the loss of spectral features associated with cytochrome
bd
. Gene disruption experiments and complementation analysis showed that the
cydC
and
cydD
gene products are required for the expression of a functional cytochrome
bd
complex. Disruption of the
cyd
genes had no apparent effect on the growth of cells in broth or defined media. The expression of the
cydABCD
operon was investigated by Northern blot analysis and by transcriptional and translational
cyd-lacZ
fusions. Northern blot analysis confirmed that
cydABCD
is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.
A Mutant of
Mycobacterium smegmatis
Defective in Dipeptide Transport
Authors: Bhatt Achal , Green Renee , Coles Roswell , Condon Michael , Connell Nancy D.
Published: December 2019

A mutant of
Mycobacterium smegmatis
unable to use the dipeptide carnosine (β-alanyl-
l
-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on β-Ala and/or
l
-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium.
CcpB, a Novel Transcription Factor Implicated in Catabolite Repression in
Bacillus subtilis
Authors: Chauvaux Sylvie , Paulsen Ian T. , Saier Milton H.
Published: December 2019

Recent work has shown that in
Bacillus subtilis
catabolite repression of several operons is mediated by a mechanism dependent on DNA-binding protein CcpA complexed to a seryl-phosphorylated derivative of HPr [HPr(Ser-P)], the small phosphocarrier protein of the phosphoenolpyruvate-sugar phosphotransferase system. In this study, it was found that a transposon insertional mutation resulted in the partial loss of gluconate (
gnt
) and xylose (
xyl
) operon catabolite repression by glucose, mannitol, and sucrose. The transposon insertion was localized to a gene, designated
ccpB
, encoding a protein 30% identical to CcpA, and relief from catabolite repression was shown to be due to the absence of CcpB rather than to the absence of a protein encoded by a downstream gene within the same operon. The relative intensities of CcpA- and CcpB-mediated catabolite repression depended on growth conditions. On solid media, and when cells were grown in liquid media with little agitation, CcpB and CcpA both proved to function in catabolite repression. However, when cells were grown in liquid media with much agitation, CcpA alone mediated catabolite repression. Like CcpA, CcpB appears to exert its catabolite-repressing effect by a mechanism dependent on the presence of HPr(Ser-P).
Bacillus subtilis
Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning
Authors: Sharpe Michaela E. , Hauser Philippe M. , Sharpe Robert G. , Errington Jeffery
Published: December 2019

Fluorescence microscopic methods have been used to characterize the cell cycle of
Bacillus subtilis
at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of
Escherichia coli
, the period required by
B. subtilis
for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of
B. subtilis
differed from that of
E. coli
in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in
E. coli
, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in
B. subtilis
should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.
Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%
Authors: Teusink Bas , Diderich Jasper A. , Westerhoff Hans V. , van Dam Karel , Walsh Michael C.
Published: December 2019

In
Saccharomyces cerevisiae
cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero
trans
-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.
Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in
Escherichia coli
Exposed to Hydrogen Peroxide
Authors: McCormick Michael L. , Buettner Garry R. , Britigan Bradley E.
Published: December 2019

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O
2
·−
and H
2
O
2
. Previous work by others has shown that
Escherichia coli
mutants lacking SOD not only are more susceptible to DNA damage and killing by H
2
O
2
but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient
E. coli
cells are exposed to increased levels of hydroxyl radical (
·
OH) as a consequence of the reaction of H
2
O
2
with this increased iron pool. When the parental
E. coli
strain AB1157 was exposed to H
2
O
2
in the presence of an α-(4-pyridyl-1-oxide)-
N
-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–
·
CH(CH
3
)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating
·
OH production. When the isogenic
E. coli
mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H
2
O
2
in a similar manner, the magnitude of
·
OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of
·
OH spin trapped. Exogenous SOD failed to inhibit
·
OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in
E. coli
SOD mutants and (ii) support the hypothesis that a resulting increase in
·
OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H
2
O
2
-mediated killing seen in these mutants lacking SOD.
Purification of
Leuconostoc mesenteroides
Citrate Lyase and Cloning and Characterization of the
citCDEFG
Gene Cluster
Authors: Bekal Sadjia , Van Beeumen Jozef , Samyn Bart , Garmyn Dominique , Henini Samia , Diviès Charles , Prévost Hervé
Published: December 2019

A citrate lyase (EC
4.1.3.6
) was purified 25-fold from
Leuconostoc mesenteroides
and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from
Leuconostoc mesenteroides
subsp.
cremoris
. This fragment was used as a probe for screening a
Leuconostoc
genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of
L. mesenteroides
is organized in three open reading frames,
citD
,
citE
, and
citF
, encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC
4.1.3.34
), and α (citrate:acetyl-ACP transferase; EC
2.8.3.10
). The gene (
citC
) encoding the citrate lyase ligase (EC
6.2.1.22
) was localized in the region upstream of
citD
. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of
Klebsiella pneumoniae
and
Haemophilus influenzae
. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the
citCDEFG
gene cluster in
Escherichia coli
led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in
E. coli
is not complete or not linked to the protein.
Regulation of
carAB
Expression in
Escherichia coli
Occurs in Part through UTP-Sensitive Reiterative Transcription
Authors: Han Xiaosi , Turnbough Charles L.
Published: December 2019

In
Escherichia coli
, expression of the
carAB
operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1. In this study, we show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription (i.e., repetitive addition of U residues to the 3′ end of a nascent transcript due to transcript-template slippage). In this case, reiterative transcription occurs at the end of a run of three T · A base pairs in the initially transcribed region of the
carAB
P1 promoter. The sequence of this region is 5′-GTTTGC (nontemplate strand). In the proposed regulatory mechanism, increased intracellular levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUU

n

(where
n
= 1 to >30). These transcripts are not extended downstream to include structural gene sequences. In contrast, lower levels of UTP enhance normal template-directed addition of a G residue at position 5 of the nascent transcript. This addition precludes reiterative transcription and permits normal transcript elongation capable of producing translatable
carAB
transcripts. Thus,
carAB
expression, which is necessary for pyrimidine nucleotide (and arginine) biosynthesis, increases in proportion to the cellular need for UTP. The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor.
Curli Fibers Are Highly Conserved between
Salmonella typhimurium
and
Escherichia coli
with Respect to Operon Structure and Regulation
Authors: Römling Ute , Bian Zhao , Hammar Mårten , Sierralta Walter D. , Normark Staffan
Published: December 2019

Mouse-virulent
Salmonella typhimurium
strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis,
csgBA(C)
and
csgDEFG
, from
S. typhimurium
SR-11 revealed the same gene order and flanking genes as in
Escherichia coli
. The divergence of the curli region between
S. typhimurium
and
E. coli
at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently,
S. typhimurium
genes on low-copy-number plasmids were able to complement respective
E. coli
mutants, although not always to wild-type levels.
rpoS
and
ompR
are required for transcriptional activation of (at least) the
csgD
promoter. The high degree of conservation at the protein level and the identical regulation patterns in
E. coli
and
S. typhimurium
suggest similar roles of curli fibers in the same ecological niche in the two species.
Relationships among the O-Antigen Gene Clusters of
Salmonella enterica
Groups B, D1, D2, and D3
Authors: Curd Heather , Liu Dan , Reeves Peter R.
Published: December 2019

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of
Salmonella enterica
group D3. This segment includes a novel O-antigen polymerase gene (
wzy
D3
). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like
wzy
gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original
wzy
gene of these O-antigen gene clusters.
Perturbation of Anion Balance during Inhibition of Growth ofEscherichia coliby Weak Acids
Authors: Roe Andrew J. , McLaggan Debra , Davidson Ian , O’Byrne Conor , Booth Ian R.
Published: December 2019

During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+and Na+levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.
Role of
rpoS
in Stress Survival and Virulence of
Vibrio cholerae
Authors: Yildiz Fitnat H. , Schoolnik Gary K.
Published: December 2019

Vibrio cholerae
is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by
rpoS
, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a
V. cholerae rpoS
homolog was cloned by functional complementation of an
Escherichia coli
mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported
rpoS
gene products. To determine the functional role of
rpoS
in
V. cholerae
, we inactivated
rpoS
by homologous recombination.
V. cholerae
strains lacking
rpoS
are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that
rpoS
may be required for the persistence of
V. cholerae
in aquatic habitats. In addition, the
rpoS
mutation led to reduced production or secretion of hemagglutinin/protease. However,
rpoS
is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.
The
apbE
Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in
Salmonella typhimurium
Authors: Beck Brian J. , Downs Diana M.
Published: December 2019

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in
Salmonella typhimurium
. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (
Escherichia coli
open reading frame designation
yojK
) at 49 min on the
S. typhimurium
chromosome. Two significant phenotypes associated with lesions in this locus (
apbE
) were identified. First,
apbE purF
double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine,
apbE
single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with
apbE
mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the
apbE
gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.
Coordination of Initiation of Nuclear Division and Initiation of Cell Division in
Schizosaccharomyces pombe
: Genetic Interactions of Mutations
Authors: Grallert A. , Grallert B. , Ribar B. , Sipiczki M.
Published: December 2019

sep1
+

encodes a
Schizosaccharomyces pombe
homolog of the HNF-3/forkhead family of the tissue-specific and developmental gene regulators identified in higher eukaryotes. Its mutant allele
sep1-1
causes a defect in cytokinesis and confers a mycelial morphology. Here we report on genetic interactions of
sep1-1
with the M-phase initiation mutations

wee1
−

,
cdc2-1w
, and
cdc25-22
. The double mutants

sep1-1 wee1
−

and
sep1-1 cdc2-1w
form dikaryon cells at high frequency, which is due to nuclear division in the absence of cell division. The dikaryosis is reversible and suppressible by
cdc25-22
. We propose that the genes

wee1
+

,

cdc2
+

,

cdc25
+

, and

sep1
+

form a regulatory link between the initiation of mitosis and the initiation of cell division.
A Major Outer Membrane Protein of
Rahnella aquatilis
Functions as a Porin and Root Adhesin
Authors: Achouak Wafa , Pages Jean-Marie , De Mot Rene , Molle Gerard , Heulin Thierry
Published: December 2019

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium
Rahnella aquatilis
CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60°C. Single channel experiments showed that this major OMP of
R. aquatilis
CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with
Escherichia coli
,
Klebsiella pneumoniae
,
Salmonella typhi
, and
Serratia marcescens
OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand β-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.
Regulation of Riboflavin Biosynthesis in
Bacillus subtilis
Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by
ribC
Authors: Mack Matthias , van Loon Adolphus P. G. M. , Hohmann Hans-Peter
Published: December 2019

This work shows that the
ribC
wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of
Bacillus subtilis
, as growth of a
ribC
deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this
ribC
deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a
B. subtilis ribC820
mutant strain, the corresponding
ribC820
gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in
B. subtilis
, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation
ribC820
leads to deregulation of riboflavin biosynthesis in
B. subtilis
, most likely by preventing the accumulation of the effector molecule FMN or FAD.
Isolation and Characterization of
Rhodobacter capsulatus
Mutants Affected in Cytochrome
cbb
3
Oxidase Activity
Authors: Koch Hans-Georg , Hwang Olivia , Daldal Fevzi
Published: December 2019

The facultative phototrophic bacterium
Rhodobacter capsulatus
contains only one form of cytochrome (cyt)
c
oxidase, which has recently been identified as a

cbb
3

-type cyt
c
oxidase. This is unlike other related species, such as
Rhodobacter sphaeroides
and
Paracoccus denitrificans
, which contain an additional mitochondrial-like
aa
3
-type cyt
c
oxidase. An extensive search for mutants affected in cyt
c
oxidase activity in
R. capsulatus
led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (
ccoNOQP
) of the cyt
cbb
3
oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that
ccoNOQP
in
R. capsulatus
is not flanked by the oxygen response regulator
fnr
, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt
cbb
3
oxidase, and unlike in
Bradyrhizobium japonicum
, no active CcoN-CcoO subcomplex was found in
R. capsulatus
. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to
ccoNOQP
is required neither for cyt
cbb
3
oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in
R. capsulatus
. The remaining cyt
c
oxidase-minus mutants mapped outside of
ccoNOQP
and formed four additional groups. In one of these groups, a fully assembled but inactive cyt
cbb
3
oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound
c
-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt
cbb
3
oxidase of
R. capsulatus.
IS
1549
from
Mycobacterium smegmatis
Forms Long Direct Repeats upon Insertion
Authors: Plikaytis Bonnie B. , Crawford Jack T. , Shinnick Thomas M.
Published: December 2019

A new insertion element, IS
1549
, was identified serendipitously from
Mycobacterium smegmatis
LR222 during experiments using a vector designed to detect the excision of IS
6110
from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the
aph
gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS
231
, which is a member of the IS
4
family of insertion elements. IS
1549
contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS
1549
are present in LR222, but not all
M. smegmatis
strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of
M. smegmatis
607 contain IS
1549
. None of 13 other species of
Mycobacterium
tested by PCR with two sets of primers specific for IS
1549
were positive for the expected amplified product.
Production of 1-Kestose in Transgenic Yeast Expressing a Fructosyltransferase from
Aspergillus foetidus
Authors: Rehm Jochen , Willmitzer Lothar , Heyer Arnd G.
Published: December 2019

Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from
Aspergillus foetidus
has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the
A. foetidus
1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of
Saccharomyces cerevisiae
resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.
Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive Dehalogenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane by
Sphingomonas paucimobilis
Authors: Miyauchi Keisuke , Suh Seug-Kyo , Nagata Yuji , Takagi Masamichi
Published: December 2019

Sphingomonas
(formerly
Pseudomonas
)
paucimobilis
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that γ-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117–3125, 1994). In the present study, we cloned and characterized a gene, designated
linD
, directly involved in the degradation of 2,5-DCHQ. The
linD
gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione
S
-transferase family. When LinD was overproduced in
Escherichia coli
, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the
linD
gene was induced by 2,5-DCHQ in
S. paucimobilis
UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing
E. coli
cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn
5
-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the
linD
region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of γ-HCH by
S. paucimobilis
UT26.
Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo
Authors: Kim Jinoh , Kendall Debra A.
Published: December 2019

SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in
Escherichia coli
. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.
Import and Metabolism of Glutathione by
Streptococcus mutans
Authors: Sherrill Christopher , Fahey Robert C.
Published: December 2019

Glutathione (γ-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including
Streptococcus mutans
ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by
S. mutans
33402 in 7H9 medium was shown to require glucose and to occur with an apparent

K
m

of 18 ± 5 μM. GSSG, GSH,
S
-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of γ-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the γ-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and γ-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of
d
/
l
-homocysteine, as well as GSH and γ-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when
S. mutans
was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by
S. mutans
ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (
S. agalactiae
ATCC 12927,
S. pyogenes
ATCC 8668, and
Enterococcus faecalis
ATCC 29212) imported glutathione at negligible or markedly lower rates.
Activation of
Escherichia coli
rRNA Transcription by FIS during a Growth Cycle
Authors: Appleman J. Alex , Ross Wilma , Salomon Julia , Gourse Richard L.
Published: December 2019

rRNA transcription in
Escherichia coli
is activated by the FIS protein, which binds upstream of
rrnp
1
promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to
rrn
transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of
fis
mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the
rrnBp
1
FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from
rrnBp
1
), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but
rrnp
1
transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from
rrnp
1
promoters during steady-state growth.
Purification of the Pyruvate Dehydrogenase Multienzyme Complex of
Zymomonas mobilis
and Identification and Sequence Analysis of the Corresponding Genes
Authors: Neveling Ute , Klasen Ralf , Bringer-Meyer Stephanie , Sahm Hermann
Published: December 2019

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium
Zymomonas mobilis
was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of
Z. mobilis
. The genes
pdhA
α (1,065 bp) and
pdhA
β (1,389 bp), encoding the E1α and E1β subunits of the E1 component, were located downstream of the gene encoding enolase. The
pdhB
(1,323 bp) and
lpd
(1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order
pdhB
-ORF2-
lpd
. Highest similarities of the gene products of the
pdhA
α,
pdhA
β, and
pdhB
genes were found with the corresponding enzymes of
Saccharomyces cerevisiae
and other eukaryotes. Like the dihydrolipoamide acetyltransferases of
S. cerevisiae
and numerous other organisms, the product of the
pdhB
gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.
The
bldD
Gene of
Streptomyces coelicolor
A3(2): a Regulatory Gene Involved in Morphogenesis and Antibiotic Production
Authors: Elliot Marie , Damji Farzana , Passantino Rosa , Chater Keith , Leskiw Brenda
Published: December 2019

The
bld
mutants of
Streptomyces coelicolor
A3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type
S. coelicolor
DNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known
bldD
mutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated
bldD
, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the
bldD
-containing fragment amplified from the chromosome of a
bldD
mutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of
bldD
transcript levels in the
bldD
+
and
bldD
mutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of
bldD
transcripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the
bldD
start codon and 7 nucleotides downstream from −10 and −35 sequences resembling
E. coli
-like streptomycete promoters.
The
bldB
Gene Encodes a Small Protein Required for Morphogenesis, Antibiotic Production, and Catabolite Control in
Streptomyces coelicolor
Authors: Pope Margaret K. , Green Brian , Westpheling Janet
Published: December 2019

Mutants blocked at the earliest stage of morphological development in
Streptomyces
species are called
bld
mutants. These mutants are pleiotropically defective in the initiation of development, the ability to produce antibiotics, the ability to regulate carbon utilization, and the ability to send and/or respond to extracellular signals. Here we report the identification and partial characterization of a 99-amino-acid open reading frame (ORF99) that is capable of restoring morphogenesis, antibiotic production, and catabolite control to all of the
bldB
mutants. Of the existing
bld
mutants,
bldB
is of special interest because the phenotype of this mutant is the most pleiotropic. DNA sequence analysis of ORF99 from each of the existing
bldB
mutants identified base changes either within the coding region of the predicted protein or in the regulatory region of the gene. Primer extension analysis identified an apparent transcription start site. A promoter fusion to the
xylE
reporter gene showed that expression of
bldB
is apparently temporally regulated and that the
bldB
gene product is involved in the regulation of its own expression.
Melanin Biosynthesis in
Cryptococcus neoformans
Authors: Williamson Peter R. , Wakamatsu Kazumasa , Ito Shosuke
Published: December 2019

Pigment production by
Cryptococcus neoformans
is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.
A Surface-Exposed Region of a Novel Outer Membrane Protein (P66) ofBorreliaspp. Is Variable in Size and Sequence
Authors: Bunikis Jonas , Luke Catherine J. , Bunikiene Elena , Bergström Sven , Barbour Alan G.
Published: December 2019

A model of the 66-kDa outer membrane protein (P66) of Lyme diseaseBorreliaspp. predicts a surface-exposed loop near the C terminus. This region contains an antigen commonly recognized by sera from Lyme disease patients. In the present study, this region of P66 and homologous proteins of otherBorreliaspp. were further investigated by using monoclonal antibodies, epitope mapping of P66 ofBorrelia burgdorferi, and DNA sequencing. A monoclonal antibody specific forB. burgdorferibound to the portion of P66 that was accessible to proteolysis in situ. The linear epitope for the antibody was mapped within a variable segment of the surface-exposed region. To further study this protein, the complete gene ofBorrelia hermsiifor a protein homologous to P66 was cloned. The deduced protein was 589 amino acids in length and 58% identical to P66 ofB. burgdorferi. TheB. hermsiiP66 protein was predicted to have a surface-exposed region in the same location as that ofB. burgdorferi’s P66 protein. With primers designed on the basis of conserved sequences and PCR, we identified and cloned the same regions of P66 proteins ofBorrelia turicatae,Borrelia parkeri,Borrelia coriaceae, andBorrelia anserina. The deduced protein sequences from all species demonstrated two conserved hydrophobic regions flanking a surface-exposed loop. The loop sequences were highly variable between differentBorreliaspp. in both sequence and size, varying between 35 and 45 amino acids. Although the actual function of P66 ofBorreliaspp. is unknown, the results suggest that its surface-exposed region is subject to selective pressure.
The CIRCE Element and Its Putative Repressor Control Cell Cycle Expression of the
Caulobacter crescentus groESL
Operon
Authors: Baldini Regina Lúcia , Avedissian Marcelo , Gomes Suely Lopes
Published: December 2019

The
groESL
operon is under complex regulation in
Caulobacter crescentus
. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the
groE
genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of
groESL
contains both a ς
32
-like promoter and a CIRCE element. Overexpression of
C. crescentus
ς
32
gives rise to higher levels of GroEL and increased levels of the
groESL
transcript coming from the ς
32
-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for this
cis
-acting element in the expression of
groESL
only at normal growth temperatures, with a minor effect on heat shock induction. Furthermore,
groESL-lacZ
transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an
hrcA
null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout the
Caulobacter
cell cycle. These results indicate a negative role for the
hrcA
gene product and the CIRCE element in the temporal control of the
groESL
operon.