bluuebunny/crossref_metadata_2025 huggingface dataset:
The Conjugal Intermediate of Plasmid RSF1010 Inhibits
Agrobacterium tumefaciens
Virulence and VirB-Dependent Export of VirE2
Authors: Stahl Lisa E. , Jacobs Amy , Binns Andrew N.
Published: December 2019

Agrobacterium tumefaciens
causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from
A. tumefaciens
to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between
Agrobacterium
strains or from
Agrobacterium
to plants also requires the
virB
-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of
Agrobacterium
inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with
virE
mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.
cis
-Acting Sequences Required for NtcB-Dependent, Nitrite-Responsive Positive Regulation of the Nitrate Assimilation Operon in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Maeda Shin-Ichi , Kawaguchi Yuriko , Ohe Taka-Aki , Omata Tatsuo
Published: December 2019

There are three binding sites for NtcA (
nir
I,
nir
II, and
nir
III), the global nitrogen regulator of cyanobacteria, in the DNA region between the two divergently transcribed operons (
nirA
and
nirB
operons) involved in nitrate assimilation in
Synechococcus
sp. strain PCC 7942. Using the
luxAB
reporter system, we showed that
nir
I and
nir
III, which are located 23 bp upstream from the −10 promoter element of
nirA
and
nirB
, respectively, are required for induction by nitrogen depletion of the
nirA
and
nirB
operons, respectively. The induction of
nirA
operon transcription was a prerequisite for the nitrite-responsive positive regulation of the transcription by NtcB, a LysR-type protein. The NtcA-binding site
nir
II, located in the middle of the
nirA-nirB
intergenic region, and a potential binding site for a LysR-type protein (TGCAN
5
TGCA; designated L1), located between
nir
I and
nir
II, were required for the nitrite-responsive, NtcB-dependent enhancement of
nirA
operon transcription. Although the requirement for the L1 site was consistent with the involvement of the LysR family protein NtcB in transcriptional regulation, NtcB did not bind to the
nirA
regulatory region in vitro in the presence of nitrite and NtcA, suggesting the involvement of some additional factor(s) in the regulation. An L1-like inverted repeat with the consensus sequence TGCN
7
GCA was conserved in the
nirA
promoter region of cyanobacteria, being centered at position −23 with respect to the NtcA-binding site corresponding to
nir
I, which suggested the common occurrence of nitrite-responsive regulation of the nitrate assimilation operon among cyanobacteria.
The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein
Authors: Pilsl Holger , Smajs David , Braun Volkmar
Published: December 2019

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of
Shigella boydii
was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.
The Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease
Pvu
I
Authors: Scharnagl Matthias , Richter Stefan , Hagemann Martin
Published: December 2019

By use of restriction endonucleases, the DNA of the cyanobacterium
Synechocystis
sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a
dam
-like site including N
6
-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain.
slr
0214, a
Synechocystis
gene encoding a putative methyltransferase that shows significant similarities to C
5
-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in
slr
0214 were generated by the insertion of an
aph
II gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of
Pvu
I. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of
slr
0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in
Synechocystis
. The Slr0214 protein was also overexpressed in
Escherichia coli
, and the purified protein demonstrated methyltransferase activity and specificity for
Pvu
I recognition sequences in vitro. We propose the designation
Syn
MI (
Synechocystis
methyltransferase I) for the
slr
0214-encoded enzyme.
A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in
Saccharomyces cerevisiae
Authors: Crowley James H. , Leak Frank W. , Shianna Kevin V. , Tove Shirley , Parks Leo W.
Published: December 2019

Aerobically growing wild-type strains of
Saccharomyces cerevisiae
are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated
upc2-1
, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that
upc2-1
was hypersensitive to Ca
2+
. Using resistance to Ca
2+
as a screening vehicle, we cloned
UPC2
and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in
upc2-1
gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of
upc2-1.
Fnr, NarP, and NarL Regulation of
Escherichia coli
K-12
napF
(Periplasmic Nitrate Reductase) Operon Transcription In Vitro
Authors: Darwin Andrew J. , Ziegelhoffer Eva C. , Kiley Patricia J. , Stewart Valley
Published: December 2019

The expression of several
Escherichia coli
operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the
napF
operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the
napF
control region. In addition, the in vivo effects of Fnr-positive control mutations on
napF
operon expression indicate that the
napF
promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of
napF
was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate
napF
operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
DNA Bending by AraC: a Negative Mutant
Authors: Saviola Beatrice , Seabold Robert R. , Schleif Robert F.
Published: December 2019

We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of
Escherichia coli
that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were
trans
dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the
araBAD
promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90° whereas the mutant bends DNA by a smaller amount.
Arginine Catabolism and the Arginine Succinyltransferase Pathway in
Escherichia coli
Authors: Schneider Barbara L. , Kiupakis Alexandros K. , Reitzer Lawrence J.
Published: December 2019

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in
Escherichia coli
(through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in
E. coli
and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in
E. coli
. Nitrogen limitation induced this pathway in both
E. coli
and
Klebsiella aerogenes
, but the mechanisms of activation clearly differed in these two organisms. We identified the
E. coli
gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an
astCADBE
operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of
astB
eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in
E. coli
and that at least one enzyme of this pathway contributes to ornithine catabolism.
Pas, a Novel Protein Required for Protein Secretion and Attaching and Effacing Activities of Enterohemorrhagic
Escherichia coli
Authors: Kresse Andreas U. , Schulze Kai , Deibel Christina , Ebel Frank , Rohde Manfred , Chakraborty Trinad , Guzmán Carlos A.
Published: December 2019

Enterohemorrhagic
Escherichia coli
(EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic
E. coli
. We have identified an open reading frame (ORF) which is located upstream of the
espA
,
espB
, and
espD
genes on the complementary strand and which exhibits high homology to the genes
spiB
from
Salmonella
,
yscD
from
Yersinia
, and
pscD
from
Pseudomonas
. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated
pas
(protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.
Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from
Streptomyces thioluteus
Authors: Shoun Hirofumi , Kano Mitsuyoshi , Baba Ikuko , Takaya Naoki , Matsuo Masaru
Published: December 2019

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N
2
O). As a representative of actinomycete denitrification systems, the system of
Streptomyces thioluteus
was investigated in detail.
S. thioluteus
attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N
2
O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.
Genetic Linkage and Cotransfer of a Novel,
vanB
-Containing Transposon (Tn
5382
) and a Low-Affinity Penicillin-Binding Protein 5 Gene in a Clinical Vancomycin-Resistant
Enterococcus faecium
Isolate
Authors: Carias Lenore L. , Rudin Susan D. , Donskey Curtis J. , Rice Louis B.
Published: December 2019

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn
5382
, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in
Enterococcus faecium
. Open reading frames internal to the right end of Tn
5382
and downstream of the
vanX
B
dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn
916
. The ends of Tn
5382
are also homologous to the ends of Tn
916
, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn
5382
excises to form a circular intermediate in
E. faecium
. Integration of Tn
5382
in the chromosome of
E. faecium
C68 has occurred 113 bp downstream of the stop codon for the
pbp5
gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an
E. faecium
recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn
5382
, the
pbp5
gene, and its putative repressor gene,
psr
. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for
vanB
operon spread in northeast Ohio. These results expand the known family of Tn
916
-related transposons, suggest a mechanism for
vanB
operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical
E. faecium
strains.
The
Pseudomonas aeruginosa rhlG
Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis
Authors: Campos-García Jesús , Caro Alma Delia , Nájera Rebeca , Miller-Maier Raina M. , Al-Tahhan Ragheb A. , Soberón-Chávez Gloria
Published: December 2019

A
Pseudomonas aeruginosa
gene homologous to the
fabG
gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this
fabG
homolog (herein called
rhlG
) produced no apparent effect on the growth rate and total lipid content of
P. aeruginosa
cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the
rhlG
mutation. We conclude that the
P. aeruginosa rhlG
gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of
rhlG
mRNA under different culture conditions is consistent with this conclusion.
Promoter Substitution and Deletion Analysis of Upstream Region Required for
rpoS
Translational Regulation
Authors: Cunning Christofer , Brown Larissa , Elliott Thomas
Published: December 2019

The RpoS sigma factor of enteric bacteria is required for the increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to high osmolarity. RpoS is also a virulence factor for several pathogenic species, including
Salmonella typhimurium
. The activity of RpoS is regulated at both the level of synthesis and protein turnover. Here we investigate the posttranscriptional control of RpoS synthesis by using
rpoS-lac
protein and operon fusions. Substitution of the native
rpoS
promoters with the
tac
or
lac
UV5 promoters allowed essentially normal regulation after growth into stationary phase in rich medium or after osmotic challenge. Regulation of these fusions required the function of
hfq
, encoding the RNA-binding protein host factor I (HF-I). Short deletions from the 5′ end of the
rpoS
transcript did not affect regulation very much; however, a larger deletion mutation that still retains 220 bp upstream of the
rpoS
ATG codon, including a proposed antisense element inhibitory for
rpoS
translation, was no longer regulated by HF-I. Several models for regulation of
rpoS
expression by HF-I are discussed.
Intraspecific Variation of Unusual Phospholipids from
Corynebacterium
spp. Containing a Novel Fatty Acid
Authors: Niepel Tanja , Meyer Holger , Wray Victor , Abraham Wolf-Rainer
Published: December 2019

The novel fatty acid
trans
-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as
Arthrobacter globiformis
) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of
Corynebacterium variabilis
DSM 20132
T
and DSM 20536. All three strains possessed the novel fatty acid, identifying
trans
-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of
Corynebacterium
sp. strain LMG 3820 were similar to those of the type strain but different from those of
Corynebacterium variabilis
DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132
T
and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.
Novel
Escherichia coli umuD
′ Mutants: Structure-Function Insights into SOS Mutagenesis
Authors: McLenigan Mary , Peat Thomas S. , Frank Ekaterina G. , McDonald John P. , Gonzalez Martín , Levine Arthur S. , Hendrickson Wayne A. , Woodgate Roger
Published: December 2019

Although it has been 10 years since the discovery that the
Escherichia coli
UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number
umuD
′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent
umuD
′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal
umuD1
,
umuD44
, and
umuD77
alleles into
umuD
′. All 17
umuD
′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.
Multidrug Efflux Pump AcrAB of
Salmonella typhimurium
Excretes Only Those β-Lactam Antibiotics Containing Lipophilic Side Chains
Authors: Nikaido Hiroshi , Basina Marina , Nguyen Vy , Rosenberg Emiko Y.
Published: December 2019

We found that the previously reported SS-B drug-supersusceptible mutant of
Salmonella typhimurium
(S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704–712, 1984) had a mutation in the
acrAB
operon. Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to β-lactam antibiotics in the complete absence of β-lactamase. The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump. This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump. According to this mechanism, the pump successfully excretes even those β-lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer. Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically. The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type
S. typhimurium
was previously interpreted as the result of poor diffusion across the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118–132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.
Deletion of the Alternative Sigma Factor ς
B
in
Staphylococcus aureus
Reveals Its Function as a Global Regulator of Virulence Genes
Authors: Kullik Ines , Giachino Philipp , Fuchs Thomas
Published: December 2019

A deletion of the
sigB
operon was constructed in three genetically distinct
Staphylococcus aureus
strains, and the phenotypes of the resulting mutants were analyzed. Compared to the corresponding wild-type strains, the Δ
sigB
mutants showed reduced pigmentation, accelerated sedimentation, and increased sensitivity to hydrogen peroxide during the stationary growth phase. A cytoplasmic protein missing in the Δ
sigB
mutants was identified as alkaline shock protein 23, and an extracellular protein excreted at higher levels in one of the Δ
sigB
mutants was identified as staphylococcal thermonuclease. Interestingly, most
sigB
deletion phenotypes were only seen in
S. aureus
COL and Newman and not in 8325, which was found to contain an 11-bp deletion in the regulator gene
rsbU
. Taken together, our results show that ς
B
is a global regulator which modulates the expression of several virulence factors in
S. aureus
and that laboratory strain 8325 is a ς
B
-defective mutant.
Topological Analysis of DcuA, an Anaerobic C
4
-Dicarboxylate Transporter of
Escherichia coli
Authors: Golby Paul , Kelly David J. , Guest John R. , Andrews Simon C.
Published: December 2019

Escherichia coli
possesses three independent anaerobic C
4
-dicarboxylate transport systems encoded by the
dcuA
,
dcuB
, and
dcuC
genes. The
dcuA
and
dcuB
genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470–6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a β-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the “positive-inside rule.” Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the
lac
UV5-promoter-based plasmid, pYZ4.
Transcriptional Regulation and Evolution of Lactose Genes in the Galactose-Lactose Operon of
Lactococcus lactis
NCDO2054
Authors: Vaughan Elaine E. , Pridmore R. David , Mollet Beat
Published: December 2019

The genetics of lactose utilization within the slow-lactose-fermenting
Lactococcus lactis
strain NCDO2054 was studied with respect to the organization, expression, and evolution of the
lac
genes. Initially the β-galactosidase gene (
lacZ
) was cloned by complementation of an
Escherichia coli
mutant on a 7-kb
Hpa
I fragment. Nucleotide sequence analysis of the complete fragment revealed part of a
gal-lac
operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is
galK-galT-lacA-lacZ-galE
; the
gal
genes encode enzymes of the Leloir pathway for galactose metabolism, and
lacA
encodes a galactoside acetyltransferase. The
galT
and
galE
genes of
L. lactis
LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting
L. lactis
C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening
lacA-lacZ
strain NCDO2054. Analysis of mRNA for the
gal
and
lac
genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the
lac
genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless β-glucuronidase gene (
gusA
) from
E. coli
, which identified three lactose-inducible intergenic promoters in the
gal-lac
operon. The greater similarity of the
lacA
and
lacZ
genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the
gal
genes, suggests that the genes within the
gal
operon of
L. lactis
NCDO2054 have been recently acquired. Thus, the
lacA-lacZ
genes appear to have engaged the promoters of the
gal
operon in order to direct and control their expression.
YscB of
Yersinia pestis
Functions as a Specific Chaperone for YopN
Authors: Jackson Michael W. , Day James B. , Plano Gregory V.
Published: December 2019

Following contact with a eucaryotic cell,
Yersinia
species pathogenic for humans (
Y. pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37°C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or “contact-dependent,” secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the
yscB
gene product in the regulation of Yop secretion in
Y. pestis
. A
yscB
deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of
yscB
in
trans
completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in
yopN
eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in
Y. pestis
.
Mutation of an Alternative Sigma Factor in the Cyanobacterium
Nostoc punctiforme
Results in Increased Infection of Its Symbiotic Plant Partner,
Anthoceros punctatus
Authors: Campbell Elsie Lin , Brahamsha Bianca , Meeks John C.
Published: December 2019

An alternative group 2 sigma factor was identified in the nitrogen-fixing, symbiotically competent cyanobacterium
Nostoc punctiforme
and designated
sigH
. Transcription of
sigH
was specifically induced within 1.5 h following exposure of
N. punctiforme
to its symbiotic plant partner,
Anthoceros punctatus
. A mutation in
sigH
resulted in a sixfold-higher initial infection of
A. punctatus
tissue without a parallel increase in nitrogen-fixing activity.
The Na
+
-Responsive
ntp
Operon Is Indispensable for Homeostatis of K
+
and Na
+
in
Enterococcus hirae
at Limited Proton Potential
Authors: Kawano Miyuki , Igarashi Kazuei , Kakinuma Yoshimi
Published: December 2019

Enterococcus hirae
ATCC 9790 grew well in Na
+
-deficient, low-K
+
medium, but growth was inhibited by carbonylcyanide
m
-chlorophenylhydrazone (CCCP). Growth inhibition and decrease of cellular K
+
levels in the presence of CCCP were relieved by the addition of Na
+
and a high concentration of K
+
. In contrast, in the mutant defective in Na
+
-ATPase or the NtpJ component of the KtrII K
+
uptake system, CCCP-induced growth inhibition was rescued by a high concentration of K
+
but not of Na
+
. These transporters are thus indispensable for homeostatis of K
+
and Na
+
at low proton potential.
Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from
Borrelia turicatae
, a Causative Agent of Relapsing Fever
Authors: Carlyon Jason A. , Marconi Richard T.
Published: December 2019

Borrelia turicatae
is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from
B. turicatae
and other relapsing-fever spirochetes that exhibits homology with the
rep
+
and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated
repA
, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that
repA
(or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both
Borrelia parkeri
and
B. turicatae
. Transcriptional analyses demonstrate that
repA
is expressed, albeit at low levels, during in vitro cultivation of
B. turicatae
. Transcriptional start site analysis revealed that
repA
is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of
repA
and its homologs suggest that RepA may play an important genus-wide role in the biology of the
Borrelia
.
Isolation and Characterization of High-Osmolarity-Sensitive Mutants of Fission Yeast
Authors: Aiba Hirofumi , Kawaura Ryosuke , Yamamoto Eiji , Yamada Hisami , Takegawa Kaoru , Mizuno Takeshi
Published: December 2019

For the fission yeast
Schizosaccharomyces pombe
, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (
gpd1
+
) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive
S. pombe
mutants, including
wis1
,
sty1
, and
gpd1
. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated
hos1
,
hos2
, and
hos3
(high osmolarity sensitive) mutants. One of them, the
hos1
mutant, was characterized in detail. The
hos1
mutant was demonstrated to have a mutational lesion in the known
ryh1
+
gene, which encodes a small GTP-binding protein. Disruption of the
ryh1
+
gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the Δ
ryh1
mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of
S. pombe
.
Structure and Mechanism of Action of the Protease That Degrades Small, Acid-Soluble Spore Proteins during Germination of Spores of
Bacillus
Species
Authors: Nessi Claudio , Jedrzejas Mark J. , Setlow Peter
Published: December 2019

The germination protease (GPR) of
Bacillus megaterium
initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P
46
) removes an ∼15-kDa C-terminal domain generating a 30-kDa species (P
30
) which is stable against further digestion. While P
30
is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P
46
to the active 41-kDa form of GPR (P
41
). Trypsin treatment of P
41
cleaves the same bond in the C-terminal part of the protein as is cleaved in the P
46
→P
30
conversion. While the ∼29-kDa species generated by trypsin treatment of P
41
is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P
41
which is 13°C lower than that for P
46
and the unfolding of P
41
at significantly lower concentrations of guanidine hydrochloride than for P
46
, are further evidence for a difference in tertiary structure between P
46
and P
41
, with P
46
presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P
46
and P
41
. The removal of ∼30% of P
46
or P
41
without significant loss in enzyme activity localized GPR’s catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P
41
by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.
Filamentous Bacteriophages of
Vibrio parahaemolyticus
as a Possible Clue to Genetic Transmission
Authors: Chang Bin , Taniguchi Hatsumi , Miyamoto Hiroshi , Yoshida Shin-ichi
Published: December 2019

We have previously reported the isolation and characterization of two filamentous bacteriophages of
Vibrio parahaemolyticus
, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of
Vibrio cholerae
and coliphage Ff of
Escherichia coli
, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of
V. parahaemolyticus
at the distinctive region of the phage genome and were also distributed on some plasmids of
V. parahaemolyticus
and total cellular DNAs of one
Vibrio damsela
and one nonagglutinable
Vibrio
strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of
V. parahaemolyticus
strains and of genetic transmission among strains through these filamentous phages.
Expression of the Kdp ATPase Is Consistent with Regulation by Turgor Pressure
Authors: Malli Ravi , Epstein Wolfgang
Published: December 2019

The
kdpFABC
operon of
Escherichia coli
encodes the four protein subunits of the Kdp K
+
transport system. Kdp is expressed when growth is limited by the availability of K
+
. Expression of Kdp is dependent on the products of the adjacent
kdpDE
operon, which encodes a pair of two-component regulators. Studies with
kdp-lac
fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464–468, 1981). More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H. Asha and J. Gowrishankar, J. Bacteriol. 175:4528–4537, 1993). We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured. In all cases, Kdp expression correlated with the K
+
concentration when growth began to slow. Making the reasonable but currently untestable assumptions that the reduction in growth rate by K
+
limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure.
The
Pseudomonas syringae
pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate
Authors: Charkowski Amy O. , Alfano James R. , Preston Gail , Yuan Jing , He Sheng Yang , Collmer Alan
Published: December 2019

The host-specific plant pathogen
Pseudomonas syringae
elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the
P. syringae
genome.
hrpW
was found in a region adjacent to the
hrp
cluster in
P. syringae
pv. tomato DC3000.
hrpW
encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from
Nectria haematococca
,
Erwinia carotovora
,
Erwinia chrysanthemi
, and
Bacillus subtilis
, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in
A
230
assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the
P. syringae
pv. syringae B728a
hrpW
was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the
P. syringae
pv. tomato
hrpW
were found in other
P. syringae
pathovars,
Pseudomonas viridiflava
,
Ralstonia
(
Pseudomonas
)
solanacearum
, and
Xanthomonas campestris
. Δ
hrpZ
::
nptII
or
hrpW
::ΩSp
r
P. syringae
pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a
hrpZ hrpW
double mutant. These features of
hrpW
and its product suggest that
P. syringae
produces multiple harpins and that the target of these proteins is in the plant cell wall.
Ndd, the Bacteriophage T4 Protein That Disrupts the
Escherichia coli
Nucleoid, Has a DNA Binding Activity
Authors: Bouet Jean-Yves , Krisch Henry M. , Louarn Jean-Michel
Published: December 2019

Early in a bacteriophage T4 infection, the phage
ndd
gene causes the rapid destruction of the structure of the
Escherichia coli
nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected
E. coli
, overexpression of the cloned
ndd
gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.
Cell Division Inhibition in
Salmonella typhimurium
Histidine-Constitutive Strains: an
ftsI
-Like Defect in the Presence of Wild-Type Penicillin-Binding Protein 3 Levels
Authors: Cano David A. , Mouslim Chakib , Ayala Juan A. , García-del Portillo Francisco , Casadesús Josep
Published: December 2019

Histidine-constitutive (His
c
) strains of
Salmonella typhimurium
undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by His
c
strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of His
c
strains. However, the His
c
septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out.
d
-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of His
c
strains may be caused by scarcity of PBP3 substrate.
Regulation of
Escherichia coli secA
by Cellular Protein Secretion Proficiency Requires an Intact Gene X Signal Sequence and an Active Translocon
Authors: Oliver Donald , Norman Jessica , Sarker Shameema
Published: December 2019

secA
is translationally regulated by the protein secretion proficiency state of the
Escherichia coli
cell. This regulation was explored by making signal sequence mutations in the gene upstream of
secA
, gene X, which promotes
secA
translational coupling. Gene X signal sequence mutants were constitutive for
secA
expression, while
prlA
alleles partially restored
secA
regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper
secA
regulation. Furthermore, gene X signal sequence mutations disrupted
secA
regulation only in the
cis
configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.
Suppression of the
ptsH
Mutation in
Escherichia coli
and
Salmonella typhimurium
by a DNA Fragment from
Lactobacillus casei
Authors: Monedero Vicente , Postma Pieter W. , Pérez-Martínez Gaspar
Published: December 2019

A DNA fragment from
Lactobacillus casei
that restores growth to
Escherichia coli
and
Salmonella typhimurium ptsH
mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of
ptsH
homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the
ptsH
phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.