bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of the Role of the Transfer Gene,
traN
, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation
Authors: Klimke William A. , Frost Laura S.
Published: December 2019

Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the
traN
gene on an F plasmid derivative, pOX38, to produce pOX38
N1
::CAT. pOX38
N1
::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1
traN
were capable of complementing pOX38
N1
::CAT transfer equally well when wild-type recipients were used. F
traN
, but not R100-1
traN
, supported a much lower level of transfer when there was an
ompA
mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1
traN
gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F
traN
suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.
Tetrachloroethene Dehalogenase from
Dehalospirillum multivorans
: Cloning, Sequencing of the Encoding Genes, and Expression of the
pceA
Gene in
Escherichia coli
Authors: Neumann Anke , Wohlfarth Gert , Diekert Gabriele
Published: December 2019

The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of
Dehalospirillum multivorans
were cloned and sequenced. The
pceA
gene is upstream of
pceB
and overlaps it by 4 bp. The presence of a ς70-like promoter sequence upstream of
pceA
and of a ρ-independent terminator downstream of
pceB
indicated that both genes are cotranscribed. This assumption is supported by reverse transcriptase PCR data. The
pceA
and
pceB
genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively. Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of
pceA
. The N-terminal amino acid sequence of the PCE dehalogenase isolated from
D. multivorans
was found 30 amino acids downstream of the N terminus of the deduced
pceA
product. The
pceA
gene contained a nucleotide stretch highly similar to binding motifs for two Fe
4
S
4
clusters or for one Fe
4
S
4
cluster and one Fe
3
S
4
cluster. A consensus sequence for the binding of a corrinoid was not found in
pceA
. No significant similarities to genes in the databases were detected in sequence comparisons. The
pceB
gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot. Sequence comparisons of
pceB
revealed no sequence similarities to genes present in the databases. Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare
Escherichia coli
tRNA
4
Arg
was
pceA
expressed, albeit nonfunctionally, in recombinant
E. coli
BL21 (DE3).
Gene Transfer between Related Bacteria by Electrotransformation: Mapping
Salmonella typhi
Genes in
Salmonella typhimurium
Authors: Toro Cecilia S. , Mora Guido C. , Figueroa-Bossi Nara
Published: December 2019

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in
Salmonella typhi
and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering
S. typhi
DNA into suitable
S. typhi
or
Salmonella typhimurium
recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in
S. typhi
were genetically mapped in
S. typhimurium
.
Identification of Two Genes from
Streptomyces argillaceus
Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin
Authors: Fernández Ernestina , Weißbach Ulrike , Reillo César Sánchez , Braña Alfredo F. , Méndez Carmen , Rohr Jürgen , Salas José A.
Published: December 2019

Mithramycin is an antitumor polyketide drug produced by
Streptomyces argillaceus
that contains two deoxysugar chains, a disaccharide consisting of two
d
-olivoses and a trisaccharide consisting of a
d
-olivose, a
d
-oliose, and a
d
-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb
Pst
I-
Xho
I fragment was sequenced, and two divergent genes (
mtmGI
and
mtmGII
) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by
mtmGI
and
mtmGII
are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.
Influence of Lif, the Lysostaphin Immunity Factor, on Acceptors of Surface Proteins and Cell Wall Sorting Efficiency in
Staphylococcus carnosus
Authors: Strauss Andreas , Thumm Günther , Götz Friedrich
Published: December 2019

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of
Staphylococcus simulans
was expressed in
Staphylococcus carnosus
, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.
Flk Couples
flgM
Translation to Flagellar Ring Assembly in
Salmonella typhimurium
Authors: Karlinsey Joyce E. , Tsui Ho-Ching T. , Winkler Malcolm E. , Hughes Kelly T.
Published: December 2019

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in
Salmonella typhimurium
. The FlgM protein inhibits the flagellum-specific transcription factor ς
28
in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The
flk
gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of ς
28
-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (
flgA
,
flgH
, and
flgI
) by introduction of a null mutation in the
flk
gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389–2400, 1997). In P- and L-ring mutant strains, recessive mutations in
flk
resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla
+
) strains. The reduction in intracellular FlgM levels by mutations in the
flk
gene was concomitant with a 10-fold increase in transcription of the
flgMN
operon compared to that of the isogenic
flk
+
strain, while transcription of the
flgAMN
operon was unaffected. This was true for both direct measurement of the
flgAMN
and
flgMN
mRNA transcripts by RNase T2 protection assays and for
lac
operon fusions to either the
flgAMN
or
flgMN
promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occured primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With
lacZ
translational fusions to
flgM
, a null mutation in
flk
resulted in a significant reduction of
flgM-lacZ
mRNA translation, expressed from the class 3
flgMN
promoter, in P- and L-ring mutant strains. No reduction in either
flgAMN
or
flgMN
mRNA stability was measured in the absence of Flk in Fla
+
, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the
flk
gene is only at the level of
flgM
mRNA translation.
Molecular Cloning and Expression Analysis of the
Rhodobacter capsulatus sodB
Gene, Encoding an Iron Superoxide Dismutase
Authors: Cortez Néstor , Carrillo Néstor , Pasternak Cécile , Balzer Angelika , Klug Gabriele
Published: December 2019

Genetic complementation of a
sodA sodB Escherichia coli
mutant strain was used to clone
Rhodobacter capsulatus
genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The
R. capsulatus sodB
gene was expressed in
E. coli
, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared
Rhodobacter
lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The
sodB
gene was expressed at low levels in
R. capsulatus
cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a
sodB
mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated
sodB
gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of
R. capsulatus
in aerobic cultures.
Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in
Pseudomonas aeruginosa
Authors: Evans Kelly , Passador Luciano , Srikumar Ramakrishnan , Tsang Eric , Nezezon Jonathon , Poole Keith
Published: December 2019

Pseudomonas aeruginosa nalB
mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in
lasI
expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of
lasI
and the virulence genes.
A Novel Mechanism for Resistance to the Antimetabolite N -Phosphonoacetyl-
l
-Aspartate by
Helicobacter pylori
Authors: Burns Brendan P. , Mendz George L. , Hazell Stuart L.
Published: December 2019

The mechanism of resistance to
N
-phosphonoacetyl-
l
-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in
Helicobacter pylori
was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of
H. pylori
. The inhibitor was taken up by
H. pylori
cells and the transport was saturable, with a

K
m

of 14.8 mM and a
V
max
of 19.1 nmol min
−1
μl of cell water
−1
. By
31
P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of
H. pylori
studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by
1
H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when
N
-acetyl-
l
-aspartate was incubated with
H. pylori
lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two
H. pylori
enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.
Posttranscriptional Control Mediates Cell Type-Specific Localization of Catalase A during
Aspergillus nidulans
Development
Authors: Navarro Rosa E. , Aguirre Jesús
Published: December 2019

Two differentially regulated catalase genes have been identified in the fungus
Aspergillus nidulans
. The
catA
gene belongs to a class whose transcripts are specifically induced during asexual sporulation (conidiation) and encodes a catalase accumulated in conidia. Using a developmental mutant affected in the
brlA
gene, which is unable to form conidia but capable of producing sexual spores (ascospores), we demonstrated that the
catA
mRNA accumulated during induction of conidiation but did not produce CatA protein. In contrast, high levels of catalase A activity were detected in the ascospores produced by this mutant, indicating that the
catA
gene is posttranscriptionally regulated. The same type of regulation was observed for a
catA
::
lacZ
translational gene fusion, suggesting that the
catA
message 5′ untranslated region could be involved in translational control during development. In a wild-type strain, β-galactosidase activity driven from the
catA
::
lacZ
gene fusion was low in hyphae and increased 50-fold during conidiation and 620-fold in isolated conidia. Consistent with this finding spatial expression of the reporter gene was restricted to metulae, phialides, and conidia. Conidium-associated expression was maintained in a
stuA
mutant, in which the conidiophore cell pattern is severely deranged.
catA
mRNA accumulation was also observed when vegetative mycelia was subject to oxidative, osmotic, and nitrogen or carbon starvation stress. Nevertheless, catalase A activity was restricted to the conidia produced under nutrient starvation. Our results provide support for a model in which translation of the
catA
message, accumulated during conidiation or in response to different types of stress, is linked to the morphogenetic processes involved in asexual and sexual spore formation. Our findings also indicate that
brlA
-independent mechanisms regulate the expression of genes encoding spore-specific products.
Haemophilus ducreyi
Secretes a Filamentous Hemagglutinin-Like Protein
Authors: Ward Christine K. , Lumbley Sheryl R. , Latimer Jo L. , Cope Leslie D. , Hansen Eric J.
Published: December 2019

We have identified two extremely large open reading frames (ORFs) in
Haemophilus ducreyi
35000,
lspA1
and
lspA2
, each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of
Bordetella pertussis
filamentous hemagglutinin (FhaB). To the best of our knowledge,
lspA1
(12,500 nucleotides [nt]) and
lspA2
(14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that
lspA1
and
lspA2
sequences were present in 15 other geographically diverse
H. ducreyi
strains. Reverse transcriptase PCR analysis of total RNA isolated from
H. ducreyi
35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of
H. ducreyi
-infected rabbits indicated that
lspA1
and
lspA2
were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulent
H. ducreyi
strains reacted with both polyclonal serum from rabbits infected with
H. ducreyi
35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in
H. ducreyi
35000 culture supernatant was shown to be the protein product of the
lspA1
ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four
H. ducreyi
strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of
H. ducreyi
to cause lesions in this animal model.
A Promoter Relay Mechanism for Sequential Gene Activation
Authors: Fang Ming , Wu Hai-Young
Published: December 2019

The effect of DNA supercoiling on gene expression is dependent not only on specific genes but also on the sequence context of the genes. This position-dependent supercoiling effect on gene activation is best illustrated in the study of the suppression of the
leu-500
mutation of the
leuABCD
operon in a
Salmonella typhimurium topA
mutant. In this communication, we report a novel promoter relay mechanism whereby several genes are sequentially expressed in a position-dependent manner: the
ilvIH
promoter (
pilvIH
) activates a cryptic
leuO
promoter (
pleuO
) located between the two divergently arrayed
ilvIH
and
leu-500
promoters. Both the
cis
-acting
pleuO
activity and the
trans
-acting LeuO protein are necessary for subsequent activation of the
leu-500
promoter (
pleu-500
). Furthermore,
pleuO
can be functionally replaced with the inducible
tac
promoter (
ptac
) for
leu-500
activation, suggesting that transcription-driven DNA supercoiling underlies the relay mechanism. This is the first example of several related genes communicating via a promoter relay mechanism for their coordinated expression.
Constricted Flux through the Branched-Chain Amino Acid Biosynthetic Enzyme Acetolactate Synthase Triggers Elevated Expression of Genes Regulated by
rpoS
and Internal Acidification
Authors: Van Dyk Tina K. , Ayers Brenda L. , Morgan Robin W. , Larossa Robert A.
Published: December 2019

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism,
Escherichia coli
genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random
E. coli
DNA fragments to
Photorhabdus luminescens luxCDABE
were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-
lux
fusions were identified. The
lux
reporter genes were joined to seven uncharacterized open reading frames,
f253a
,
f415
,
frvX
,
o513
,
o521
,
yciG
, and
yohF
, and five known genes,
inaA
,
ldcC
,
osmY
,
poxB
, and
sohA
. Inactivation of the
rpoS
-encoded sigma factor, ς
S
, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the ς
S
regulon,
f253a
,
ldcC
,
yciG
, and
yohF
, and included two known members,
osmY
and
poxB
. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-
lux
fusion-containing strains, namely, those with fusions of the ς
S
-controlled genes and
inaA
. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the ς
S
-dependent stress response.
Molecular Cloning and Characterization of Fengycin Synthetase Gene
fenB
from
Bacillus subtilis
Authors: Lin Guang-Huey , Chen Chyi-Liang , Tschen Johannes Scheng-Ming , Tsay San-San , Chang Yu-Sun , Liu Shih-Tung
Published: December 2019

A fengycin synthetase gene,
fenB
, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in
Escherichia coli
and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, a

K
m

value of 922 μM, and a turnover number of 236 s
−1
. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.
Iron Uptake inUstilago maydis: Tracking the Iron Path
Authors: Ardon Orly , Nudelman Raphael , Caris Catherine , Libman Jacqueline , Shanzer Abraham , Chen Yona , Hadar Yitzhak
Published: December 2019

In this study, we monitored and compared the uptake of iron in the fungusUstilago maydisby using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.
FtsZ Dynamics during the Division Cycle of Live
Escherichia coli
Cells
Authors: Sun Qin , Margolin William
Published: December 2019

The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing
Escherichia coli
cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.
Mosaic Structure of the
smpB-nrdE
Intergenic Region of
Salmonella enterica
Authors: Bäumler Andreas J. , Heffron Fred
Published: December 2019

The
Salmonella enterica smpB-nrdE
intergenic region contains about 45 kb of DNA that is not present in
Escherichia coli
. This DNA region was not introduced by a single horizontal transfer event, but was generated by multiple insertions and/or deletions that gave rise to a mosaic structure in this area of the chromosome.
The 102-Kilobase Unstable Region of
Yersinia pestis
Comprises a High-Pathogenicity Island Linked to a Pigmentation Segment Which Undergoes Internal Rearrangement
Authors: Buchrieser Carmen , Prentice Michael , Carniel Elisabeth
Published: December 2019

Several pathogenicity islands have recently been identified in different bacterial species, including a high-pathogenicity island (HPI) in
Yersinia enterocolitica
1B. In
Y. pestis
, a 102-kb chromosomal fragment (
pgm
locus) that carries genes involved in iron acquisition and colony pigmentation can be deleted en bloc. In this study, characterization and mapping of the 102-kb region of
Y. pestis
6/69 were performed to determine if this unstable region is a pathogenicity island. We found that the 102-kb region of
Y. pestis
is composed of two clearly distinct regions: an ≈35-kb iron acquisition segment, which is an HPI per se, linked to an ≈68-kb pigmentation segment. This linkage was preserved in all of the
Y. pestis
strains studied. However, several nonpigmented
Y. pestis
strains harboring an
irp2
gene have been previously identified, suggesting that the pigmentation segment is independently mobile. Comparison of the physical map of the 102-kb region of these strains with that of strain 6/69 and complementation experiments were carried out to determine the genetic basis of this phenomenon. We demonstrate that several different mechanisms involving mutations and various-size deletions are responsible for the nonpigmented phenotype in the nine strains studied. However, no deletion corresponded exactly to the pigmentation segment. The 102-kb region of
Y. pestis
is an evolutionarily stable linkage of an HPI with a pigmentation segment in a region of the chromosome prone to rearrangement in vitro.
Ferric Citrate Transport of
Escherichia coli
: Functional Regions of the FecR Transmembrane Regulatory Protein
Authors: Welz Dietrich , Braun Volkmar
Published: December 2019

Transcription of the ferric citrate transport genes of
Escherichia coli
is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM β-lactamase. BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance. Cells that synthesized FecR′-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin. The ampicillin resistance of the low-resistance FecR′-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in
degP
and
ompT
protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation. Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the β-lactamase activity of high-resistance FecR′-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence. All FecR′-BlaM proteins with at least 61 FecR residues complemented a
fecR
mutant such that it could grow on ferric citrate as the sole iron source and induced
fecA-lacZ
transcription independent of ferric citrate. The low resistance mediated by two FecR′-BlaM proteins in a
fecA
deletion mutant was increased 20-fold by transformation with a
fecA
-encoding plasmid. We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.
The Prosequence of Pro-ς
K
Promotes Membrane Association and Inhibits RNA Polymerase Core Binding
Authors: Zhang Bin , Hofmeister Antje , Kroos Lee
Published: December 2019

Pro-ς
K
is the inactive precursor of ς
K
, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of
Bacillus subtilis
. Upon subcellular fractionation, the majority of the pro-ς
K
was present in the membrane fraction. The rest of the pro-ς
K
was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ς
K
was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-ς
E
was analyzed. Pro-ς
K
was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-ς
K
did not require
spoIVF
gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-ς
K
processing in the mother cell. Furthermore, pro-ς
K
associated with the membrane when overproduced in vegetative cells. Overproduction of pro-ς
K
in sporulating cells resulted in more pro-ς
K
in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-ς
K
was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-ς
K
. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-ς
K
and ς
K
from other cell constituents. After salt removal, ς
K
, but not pro-ς
K
, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-ς
K
to the membrane, where it may interact with the processing machinery.
Purification, Properties, and Characterization of Recombinant
Streptomyces
sp. Strain C5 DoxA, a Cytochrome P-450 Catalyzing Multiple Steps in Doxorubicin Biosynthesis
Authors: Walczak Robbie J. , Dickens Michael L. , Priestley Nigel D. , Strohl William R.
Published: December 2019

DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from
Streptomyces lividans
transformed with a plasmid containing the
Streptomyces
sp. strain C5
doxA
gene under the control of the strong SnpR-activated
snpA
promoter. The purified enzyme was a monomeric, soluble protein with an apparent
M
r
of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30°C. The
k
cat
/

K
m

values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M
−1
· s
−1
; for 13-dihydrodaunorubicin, 14,000 M
−1
· s
−1
; for 13-dihydrocarminomycin, 280 M
−1
· s
−1
; and for daunorubicin, 130 M
−1
· s
−1
. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-
O
-methyl series of anthracyclines.
( R )-Citramalate Synthase in Methanogenic Archaea
Authors: Howell David M. , Xu Huimin , White Robert H.
Published: December 2019

The
Methanococcus jannaschii
gene MJ1392 was cloned, and its protein product was hyperexpressed in
Escherichia coli
. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (
R
)-citramalate. Thus, this gene (
cimA
) encodes an (
R
)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine.
Septal Localization of Penicillin-Binding Protein 1 in
Bacillus subtilis
Authors: Pedersen Lotte B. , Angert Esther R. , Setlow Peter
Published: December 2019

Previous studies have shown that
Bacillus subtilis
cells lacking penicillin-binding protein 1 (PBP1), encoded by
ponA
, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg
2+
, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of
B. subtilis
. In addition, we have used fluorescence and electron microscopy to show that growing
ponA
mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most
ponA
mutant cells, a significant proportion of
ponA
mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in
B. subtilis
. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.
Function of Proline Residues of MotA in Torque Generation by the Flagellar Motor of
Escherichia coli
Authors: Braun Timothy F. , Poulson Susan , Gully Jonathan B. , Empey J. Courtney , Van Way Susan , Putnam Angélica , Blair David F.
Published: December 2019

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from
Escherichia coli
) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D
2
O versus H
2
O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.
Membrane Topology of MntB, the Transmembrane Protein Component of an ABC Transporter System for Manganese in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Bartsevich Victor V. , Pakrasi Himadri B.
Published: December 2019

The structure of the membrane protein MntB, a component of a manganese transporter system in
Synechocystis
sp. strain PCC 6803, was examined with a series of fusions to the reporter proteins alkaline phosphatase and β-galactosidase. The results support a topological model for MntB consisting of nine transmembrane segments, with the amino terminus of the protein being in the periplasm and the carboxyl terminus being in the cytoplasm.
Increased
rrn
Gene Dosage Causes Intermittent Transcription of rRNA in
Escherichia coli
Authors: Voulgaris Justina , French Sarah , Gourse Richard L. , Squires Craig , Squires Catherine L.
Published: December 2019

When the number of rRNA (
rrn
) operons in an
Escherichia coli
cells is increased by adding an
rrn
operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell. We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carrying
rrnB
. We found that there were fewer RNA polymerase molecules transcribing the
rrn
genes, as predicted from previous gene dosage studies. Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon. No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed. We also found that the overall transcription elongation rate was unchanged when the
rrn
gene dosage was increased. Our data suggest that when
rrn
gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals.
The
Yersinia enterocolitica
pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C
Authors: Rohde John R. , Luan Xing-she , Rohde Harold , Fox James M. , Minnich S. A.
Published: December 2019

Temperature has a pleiotropic effect on
Yersinia enterocolitica
gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (≤30°C) and virulence plasmid-encoded Yop secretion (37°C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the
Y. enterocolitica
virulence plasmid, pYV, undergoes a conformational transition between 30 and 37°C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including
virF
, the positive activator of virulence genes. These bends are detectable at 30°C but melt at 37°C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from
Y. enterocolitica
were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the
Y. enterocolitica
pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in
Y. enterocolitica
and other enteric pathogens.
Glucose Transport in the Extremely Thermoacidophilic
Sulfolobus solfataricus
Involves a High-Affinity Membrane-Integrated Binding Protein
Authors: Albers Sonja-V. , Elferink Marieke G. L. , Charlebois Robert L. , Sensen Christoph W. , Driessen Arnold J. M. , Konings Wil N.
Published: December 2019

The archaeon
Sulfolobus solfataricus
grows optimally at 80°C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (

K
d

of 0.43 μM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the
S. solfataricus
P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in
Pyrococcus horikoshii
OT3 was found to be organized in an operon. These data indicate that
S. solfataricus
utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.
Regulation of Gene Expression by Glucose inSaccharomyces cerevisiae: a Role for ADA2and ADA3/NGG1
Authors: Wu Mei , Newcomb Laura , Heideman Warren
Published: December 2019

WhenSaccharomyces cerevisiaecells are transferred from poor medium to fresh medium containing glucose, they rapidly increase the transcription of a large group of genes as they resume rapid growth and accelerate progress through the cell cycle. Among those genes induced by glucose isCLN3, encoding a G1cyclin that is thought to play a pivotal role in progression through Start. Deletion ofCLN3delays the increase in proliferation normally observed in response to glucose medium.ADA2andADA3/NGG1are necessary for the rapid induction ofCLN3message levels in response to glucose. Loss of eitherADA2orADA3/NGG1also affects a large number of genes and inhibits the rapid global increase in transcription that occurs in response to glucose. Surprisingly, these effects are transitory, and expression ofCLN3and total poly(A)+RNA appear normal whenADA2orADA3/NGG1deletion mutants are examined in log-phase growth. These results indicate a role forADA2andADA3/NGG1in allowing rapid transcriptional responses to environmental signals. Consistent with the role of the Ada proteins in positive regulation ofCLN3, deletion ofRPD3, encoding a histone deacetylase, prevented the down regulation ofCLN3mRNA in the absence of glucose.
Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange
Authors: Pavelka Martin S. , Jacobs William R.
Published: December 2019

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organismsMycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains ofM. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organismMycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.
Moraxella (Branhamella)catarrhalis BRO β-Lactamase: a Lipoprotein of Gram-Positive Origin?
Authors: Bootsma Hester J. , Aerts Piet C. , Posthuma George , Harmsen Theo , Verhoef Jan , van Dijk Hans , Mooi Frits R.
Published: December 2019

In the past 20 years, BRO β-lactamase-producingMoraxella catarrhalisstrains have emerged. We show that BRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To our knowledge, this is the first description of a lipidated β-lactamase in a gram-negative species.
Escherichia coli Gene ydeA Encodes a Major Facilitator Pump Which Exports l-Arabinose and Isopropyl-β-d-Thiogalactopyranoside
Authors: Carolé Sandra , Pichoff Sébastien , Bouché Jean-Pierre
Published: December 2019

Inactivation of the Escherichia coli geneydeA, which encodes a member of the major facilitator superfamily, decreased the efflux of l-arabinose, thereby affecting the expression of AraC-regulated genes. In addition, overexpression of ydeA decreased the expression of genes regulated by isopropyl-β-d-thiogalactopyranoside.