bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological States of Individual
Salmonella typhimurium
Cells Monitored by In Situ Reverse Transcription-PCR
Authors: Holmstrøm Kim , Tolker-Nielsen Tim , Molin Søren
Published: December 2019

The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of
groEL
and
tsf
mRNAs per cell in
Salmonella typhimurium
cells in different physiological states were obtained by Northern analysis. The average number of
groEL
mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures. The average number of
tsf
mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures. The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of
groEL
and
tsf
mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed. Neither
groEL
nor
tsf
mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR. The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to
groEL
mRNA single-cell contents, suggesting a cell-cycle-controlled expression of
groEL
in
S. typhimurium
, whereas a fast-growing culture is homogeneous with respect to
tsf
mRNA single-cell contents, suggesting that the level of
tsf
mRNA is relatively constant during the cell cycle.
Functional Identification of the Product of the
Bacillus subtilis yvaL
Gene as a SecG Homologue
Authors: van Wely Karel H. M. , Swaving Jelto , Broekhuizen Cees P. , Rose Matthias , Quax Wim J. , Driessen Arnold J. M.
Published: December 2019

Protein export in
Escherichia coli
is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain. In the gram-positive bacterium
Bacillus subtilis
, SecA, SecY, and SecE have been identified through genetic analysis. Sequence comparison of the
Bacillus
chromosome identified a potential homologue of SecG, termed YvaL. A chromosomal disruption of the
yvaL
gene results in mild cold sensitivity and causes a β-lactamase secretion defect. The cold sensitivity is exacerbated by overexpression of the secretory protein α-amylase, whereas growth and β-lactamase secretion are restored by coexpression of
yvaL
or the
E. coli secG
gene. These results indicate that the
yvaL
gene codes for a protein that is functionally homologous to SecG.
Role of ArgR in Activation of the
ast
Operon, Encoding Enzymes of the Arginine Succinyltransferase Pathway in
Salmonella typhimurium
Authors: Lu Chung-Dar , Abdelal Ahmed T.
Published: December 2019

The
ast
operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from
Salmonella typhimurium
, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an
argR
derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the
ast
operon in response to nitrogen or carbon limitation.
Mutational Analysis of the
phoD
Promoter in
Bacillus subtilis
: Implications for PhoP Binding and Promoter Activation of Pho Regulon Promoters
Authors: Eder Steve , Liu Wei , Hulett F. Marion
Published: December 2019

The PhoP-PhoR two-component regulatory system controls the phosphate deficiency response in
B. subtilis
. A number of Pho regulon genes which require PhoP∼P for activation or repression have been identified. The studies reported here were initiated to understand the PhoP-DNA interaction necessary for Pho promoter regulation. The regulatory region of
phoD
was characterized in detail using oligo-directed mutagenesis, DNase I footprinting, and in vivo transcription assays. These data reveal basic principles of PhoP binding relevant to PhoP’s interaction with other Pho regulon promoters. Our results show that: (i) a dimer of PhoP∼P is able to bind two consensus repeats in a stable fashion; (ii) PhoP binding is highly cooperative within the core promoter region, which is located from −66 to −17 on the coding strand and contains four TT(A/T/C)ACA-like repeats; (iii) specific bases comprising the TT(A/T/C)ACA consensus are essential for transcriptional activation, but the specific base pairs of the intervening sequences separating the consensus repeats are not important for either PhoP binding or promoter activation; (iv) the spacing between two consensus repeats within a putative dimer binding site in the core region is important for both PhoP binding and promoter activation; (v) the exact spacing between two dimer binding sites within the core region is important for promoter activation but less so for PhoP binding affinity, as long as the repeats are on the same face of the helix; and (vi) the 5′ secondary binding region is important for coordinated PhoP binding to the core binding region, making it nearly essential for promoter activation.
Role of BkdR, a Transcriptional Activator of the SigL-Dependent Isoleucine and Valine Degradation Pathway in
Bacillus subtilis
Authors: Debarbouille Michel , Gardan Rozenn , Arnaud Maryvonne , Rapoport George
Published: December 2019

A new gene,
bkdR
(formerly called
yqiR
), encoding a regulator with a central (catalytic) domain was found in
Bacillus subtilis
. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called
yqiS
,
yqiT
,
yqiU
,
yqiV
,
bfmBAA
,
bfmBAB
, and
bfmBB
and now referred to as
ptb
,
bcd
,
buk
,
lpd
,
bkdA1
,
bkdA2
, and
bkdB
, are located downstream from the
bkdR
gene in
B. subtilis
. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α (dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in
bcd
and
bkdR
null mutants of
B. subtilis
. The seven genes appear to be organized as an operon,
bkd
, transcribed from a −12, −24 promoter. The expression of the
bkd
operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene
bkdR
. Deletion analysis showed that upstream activating sequences are involved in the expression of the
bkd
operon and are probably the target of
bkdR
. Transcription of the
bkd
operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.
Analysis of the Role of
trans
-Translation in the Requirement of tmRNA for λ
imm
P22
Growth in
Escherichia coli
Authors: Withey Jeffrey , Friedman David
Published: December 2019

The small, stable RNA molecule encoded by
ssrA
, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid λ
imm
P22
phages in
Escherichia coli
. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, the
trans
-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990–993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on λ
imm
P22
growth of mutations eliminating activities postulated to be important for two different steps in the
trans
-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for λ
imm
P22
growth in
E. coli
, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that
trans
-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation.
Transcriptional Activation of
ydeA
, Which Encodes a Member of the Major Facilitator Superfamily, Interferes with Arabinose Accumulation and Induction of the
Escherichia coli
Arabinose P
BAD
Promoter
Authors: Bost Sandrine , Silva Filo , Belin Dominique
Published: December 2019

Induction of genes expressed from the arabinose P
BAD
promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the
ydeA
promoter and stimulate
ydeA
transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient −35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The
ydeA
gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of
ydeA
has no detectable effect on cell growth. Thus,
ydeA
appears to be nonessential under usual laboratory growth conditions. The
ydeA
gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of
ydeA
transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.
Mutations That Confer Resistance to 2-Deoxyglucose Reduce the Specific Activity of Hexokinase from
Myxococcus xanthus
Authors: Youderian Philip , Lawes Matthew C. , Creighton Chad , Cook Jessica C. , Saier Milton H.
Published: December 2019

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of
Myxococcus xanthus
. Mutants of
M. xanthus
resistant to 2dGlc, designated
hex
mutants, arise at a low spontaneous frequency. Expression of the
Escherichia coli glk
(glucokinase) gene in
M. xanthus hex
mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of
M. xanthus
extracts reveal a soluble hexokinase (ATP:
d
-hexose-6-phosphotransferase; EC
2.7.1.1
) activity but no phosphotransferase system activities. The
hex
mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the
hex
mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and
N
-acetylglucosamine act as inhibitors of glucose turnover by the
M. xanthus
hexokinase in vitro, consistent with the finding that glucose and
N
-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.
Mutational Analysis of the
vacA
Promoter Provides Insight into Gene Transcription in
Helicobacter pylori
Authors: Forsyth Mark H. , Cover Timothy L.
Published: December 2019

Analysis of 12
Helicobacter pylori
promoters indicates the existence of a consensus −10 hexamer (TAtaaT) but little conservation of −35 sequences. In this study, mutations in either the
H. pylori vacA
−10 region or the −35 region resulted in decreased
vacA
transcription and suggested that an extended −10 motif is utilized. Thus, despite the lack of a −35 consensus sequence for
H. pylori
promoters, the −35 region plays a functional role in
vacA
transcription.
Isolation of an Active Catalytic Core of
Streptococcus downei
MFe28 GTF-I Glucosyltransferase
Authors: Monchois Vincent , Arguello-Morales Martha , Russell Roy R. B.
Published: December 2019

Truncated variants of GTF-I from
Streptococcus downei
MFe28 were purified by means of a histidine tag. Sequential deletions showed that the C-terminal domain was not directly involved in the catalytic process but was required for primer activation. A fully active catalytic core of only 100 kDa was isolated.
Regulated Expression of the
Streptococcus mutans dlt
Genes Correlates with Intracellular Polysaccharide Accumulation
Authors: Spatafora Grace A. , Sheets Megan , June Rebecca , Luyimbazi David , Howard Katherine , Hulbert Robin , Barnard Daron , el Janne Mariam , Hudson Michael C.
Published: December 2019

Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to
Streptococcus mutans
-induced cariogenesis. We previously identified and cloned a locus from the
S. mutans
chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among
d
-alanine-activating enzymes (DltA) in
Lactobacillus rhamnosus
(formerly
Lactobacillus casei
) and
Bacillus subtilis
. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the
S. mutans
chromosome which is separate from the
glgP
,
glgA
, and
glgD
genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a new
dlt
designation to the locus which we had formerly called
glg
. We maintain that the
dlt
genes are involved in
S. mutans
IPS accumulation, however, since they complement a mutation in
trans
which otherwise renders
S. mutans
IPS deficient. In this study, we found that expression of the
S. mutans dlt
genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the
S. mutans dlt
genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in
dlt
expression is a similar constitutive expression of the
dlt
genes in an
S. mutans
PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the
dlt
gene products in
S. mutans
IPS accumulation and suggest that
dlt
expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.
Promoter Analysis of the
cap8
Operon, Involved in Type 8 Capsular Polysaccharide Production in
Staphylococcus aureus
Authors: Ouyang Shu , Sau Subrata , Lee Chia Y.
Published: December 2019

The production of type 8 capsular polysaccharide (CP8) in
Staphylococcus aureus
is regulated in response to a variety of environmental factors. The
cap8
genes required for the CP8 production in strain Becker are transcribed as a single large transcript by a primary promoter located within a 0.45-kb region upstream of the first gene of the
cap8
gene cluster. In this study, we analyzed the primary
cap8
promoter region in detail. We determined the transcription initiation site of the primary transcript by primer extension and identified the potential promoter sequences. We found several inverted and direct repeats upstream of the promoter. Deletion analysis and site-directed mutagenesis showed that a 10-bp inverted repeat of one of the repeats was required for promoter activity. We showed that the distance but not the specific sequences between the inverted repeat and the promoter was critical to the promoter activity. However, insertion of a DNA sequence with two or four helix turns in this intervening region had a slight effect on promoter activity. To demonstrate the biological significance of the 10-bp inverted repeat, we constructed a strain with a mutation in the repeat in the
S. aureus
Becker chromosome and showed that the repeat affected CP8 production mostly at the transcriptional level. By gel mobility shift assay, we demonstrated that strain Becker produced at least one protein capable of specific binding to the 10-bp inverted repeat, indicating that the repeat serves as a positive regulatory protein binding site. In addition, reporter gene fusion analysis showed that the
cap8
promoter activity was influenced by various growth media and affected most by yeast extract. Our results suggest that yeast extract may exert its profound inhibitory effect on
cap8
gene expression through the 10-bp inverted repeat element.
The Extracellular Domain of the
Saccharomyces cerevisiae
Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity
Authors: Ostrander Darin B. , Gorman Jessica A.
Published: December 2019

The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast
Saccharomyces cerevisiae
. Truncations of
SLN1
that retain an intact kinase domain are capable of complementing the lethality of an
sln1
Δ strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various
SLN1
constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (ΔTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the ΔTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.
Clathrin and Two Components of the COPII Complex, Sec23p and Sec24p, Could Be Involved in Endocytosis of the
Saccharomyces cerevisiae
Maltose Transporter
Authors: Peñalver Élida , Lucero Pilar , Moreno Eulalia , Lagunas Rosario
Published: December 2019

The
Saccharomyces cerevisiae
maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis. Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin. In this work, we attempted to determine which coat proteins are involved in this endocytosis. Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter. The results also indicate that endocytosis of the maltose transporter and of the α-factor receptor could have different requirements.
Bacteroides fragilis
Transfer Factor Tn
5520
: the Smallest Bacterial Mobilizable Transposon Containing Single Integrase and Mobilization Genes That Function in
Escherichia coli
Authors: Vedantam Gayatri , Novicki Thomas J. , Hecht David W.
Published: December 2019

Many bacterial genera, including
Bacteroides
spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn
5520
, a new mobilizable transposon from
Bacteroides fragilis
LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn
5520
was captured from
B. fragilis
LV23 by using the transfer-deficient shuttle vector pGAT400ΔBglII. The termini of Tn
5520
contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn
5520
also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn
5520
has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn
5520
have been studied, revealing that Tn
5520
mobilizes plasmids in both
B. fragilis
and
Escherichia coli
at high frequency and also transposes in
E. coli.
cis
-Acting Elements Responsible for Low-Temperature-Inducible Expression of the Gene Coding for the Thermolabile Isocitrate Dehydrogenase Isozyme of a Psychrophilic Bacterium,
Vibrio
sp. Strain ABE-1
Authors: Sahara Takehiko , Suzuki Masahiro , Tsuruha Jun-Ichiro , Takada Yasuhiro , Fukunaga Noriyuki
Published: December 2019

Transcriptional control of the low-temperature-inducible
icdII
gene, encoding the thermolabile isocitrate dehydrogenase of a psychrophilic bacterium,
Vibrio
sp. strain ABE-1, was found to be mediated in part by a transcriptional silencer locating at nucleotide positions −560 to −526 upstream from the transcription start site of
icdII
. Deletion of the silencer resulted in a 20-fold-increased level of expression of the gene at low temperature (15°C) but not at high temperature (37°C). In addition, a CCAAT sequence located 2 bases upstream of the −35 region was found to be essential for the low-temperature-inducible expression of the gene. By deletion of this sequence, low-temperature-dependent expression of the gene was completely abolished. The ability of the
icdII
promoter to control the expression of other genes was confirmed by using a fusion gene containing the
icdII
promoter region and the promoterless
icdI
open reading frame, which encodes the non-cold-inducible isocitrate dehydrogenase isozyme of
Vibrio
sp. strain ABE-1.
Escherichia coli
transformants harboring
icdII
acquired an ability to grow rapidly at low temperature.
Acarbose, a Pseudooligosaccharide, Is Transported but Not Metabolized by the Maltose-Maltodextrin System of
Escherichia coli
Authors: Brunkhorst Claudia , Andersen Christian , Schneider Erwin
Published: December 2019

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of
Escherichia coli
. Acarbose at millimolar concentrations specifically affected the growth of
E. coli
K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a

K
i

of 1.1 μM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).
Differential Regulation of Two Divergent
Sinorhizobium meliloti
Genes for HPII-Like Catalases during Free-Living Growth and Protective Role of Both Catalases during Symbiosis
Authors: Sigaud Samuel , Becquet Vanessa , Frendo Pierre , Puppo Alain , Hérouart Didier
Published: December 2019

Two catalases, KatA and KatB, have been detected in
Sinorhizobium meliloti
growing on rich medium. Here we characterize a new catalase gene encoding a third catalase (KatC). KatC activity was detectable only at the end of the stationary phase in
S. meliloti
growing in minimum medium, whereas KatA activity was found during the exponential phase. Analysis with a
katC-lacZ
fusion demonstrated that
katC
expression is mainly regulated at the transcription level. An increase of catalase activity correlating with KatA induction was detected in bacteroids. A dramatic decrease of nitrogen fixation capacity in a
katA katC
double mutant was observed, suggesting that these catalases are very important for the protection of the nitrogen fixation process.
A Novel Aromatic-Ring-Hydroxylating Dioxygenase from the Diterpenoid-Degrading Bacterium
Pseudomonas abietaniphila
BKME-9
Authors: Martin Vincent J. J. , Mohn William W.
Published: December 2019

Pseudomonas abietaniphila
BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The
ditA1
,
ditA2
, and
ditA3
genes, which encode the α and β subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9.2 kb upstream of the oxygenase genes and 872 bp upstream of a putative
meta
ring cleavage dioxygenase gene,
ditC
. A Tn
5
insertion in the α subunit gene,
ditA1
, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene,
ditA3
, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in
Escherichia coli
of
ditA1A2A3
, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11,12-dihydroxy-8,13-abietadien acid, which was identified by
1
H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the α subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.
Glycosyltransferase Domain of Penicillin-Binding Protein 2a from
Streptococcus pneumoniae
Is Membrane Associated
Authors: di Guilmi Anne Marie , Mouz Nicolas , Martin Lydie , Hoskins JoAnn , Jaskunas S. Richard , Dideberg Otto , Vernet Thierry
Published: December 2019

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to β-lactams in
Streptococcus pneumoniae
is caused by low-affinity PBPs.
S. pneumoniae
PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of
S. pneumoniae
PBP 2a has been expressed (PBP 2a*) in
Escherichia coli
as a glutathione
S
-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for β-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other β-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
Requirement of NifX and Other
nif
Proteins for In Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase
Authors: Shah Vinod K. , Rangaraj Priya , Chatterjee Ranjini , Allen Ronda M. , Roll Jon T. , Roberts Gary P. , Ludden Paul W.
Published: December 2019

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of
Azotobacter vinelandii
defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of
A. vinelandii
DJ42.48 (Δ
nifENX ΔvnfE
) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.
The
Staphylococcus aureus rsbW
(
orf159
) Gene Encodes an Anti-Sigma Factor of SigB
Authors: Miyazaki Eishi , Chen Jong-Min , Ko Chiew , Bishai William R.
Published: December 2019

SigB, a newly discovered alternative sigma factor of
Staphylococcus aureus
, has been shown to play an important role in stress responses and the regulation of virulence factors. The
rsbW
(
orf159
) gene is immediately upstream of
sigB
. Its gene product is homologous to
Bacillus subtilis
RsbW which under appropriate conditions binds to
B. subtilis
SigB and functions as an anti-sigma factor or negative posttranslational regulator. To define the function of
S. aureus
RsbW, both the
S. aureus
SigB and RsbW proteins were expressed in
Escherichia coli
and purified. Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB. In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the
sar
P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent. We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro. These results show that RsbW is a protein sequestering anti-sigma factor of
S. aureus
SigB and suggest that SigB activity in
S. aureus
is regulated posttranslationally.
Virulence Factors of
Escherichia coli
Isolates From Patients with Symptomatic and Asymptomatic Bacteriuria and Neuropathic Bladders Due to Spinal Cord and Brain Injuries
Authors: Hull Richard A. , Rudy Delbert C. , Wieser Inge E. , Donovan William H.
Published: December 2019

Chronic bacteriuria is a common occurrence among spinal-cord injury patients and others with neuropathic bladders. If bacteria are present in the urinary tract, the patient may develop symptoms of infection or remain asymptomatic. We have compared virulence properties of 28
Escherichia coli
isolates from patients with symptomatic urinary tract infections (UTI) and 29
E. coli
isolates from patients with asymptomatic bacteriuria (ABU). Bacteria from patients with symptomatic UTI were more likely to be hemolytic than isolates from patients with ABU (
P
= 0.05) or fecal isolates obtained from healthy volunteers (
P
< 0.001). Bacteria from patients with symptomatic UTI were also more likely than strains isolated from patients with ABU (
P
= 0.08) or fecal strains (
P
< 0.001) to exhibit
d
-mannose-resistant hemagglutination of human erythrocytes. The results suggest that
E. coli
isolates from nonimmunocompromised patients who require intermittent catheterization and who develop symptomatic UTI may be distinguished from bacteria recovered from patients who remain asymptomatic and possibly from normal fecal
E. coli.
Clinical Application of PCR-Restriction Enzyme Pattern Analysis for Rapid Identification of Aerobic Actinomycete Isolates
Authors: Wilson Rebecca W. , Steingrube Vincent A. , Brown Barbara A. , Wallace Richard J.
Published: December 2019

The accuracy and practicality of PCR-restriction enzyme pattern analysis (PRA) for routine identification of aerobic actinomycete clinical isolates were evaluated for 299 cultures submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler. PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by traditional methods, including growth characteristics, susceptibility patterns, biochemical testing, and high-performance liquid chromatography analysis. Microbiological examination of six cultures ruled out aerobic actinomycetes, and they were omitted from the study. Amplicons were analyzed with
Bst
EII,
Hae
III,
Msp
I,
Hin
fI, and
Bsa
HI. When necessary,
Aci
I,
Hha
I, and
Nar
I were also used. From March 1995 through May 1997 (27 months), 274 of the remaining 293 (93.5%) isolates were accurately identified by PRA. Major diagnostic groups included 170 mycobacteria, 93 nocardiae, and 30 other aerobic actinomycetes. Mixed cultures were readily recognized by PRA, including a wound culture that contained two
Nocardia
taxa that were indistinguishable morphologically.
Mycobacterium mucogenicum
was identified in three cultures heavily contaminated with gram-positive cocci. The 19 isolates that produced PRA patterns that did not match those in the current PRA database were differentiated into 8
Mycobacterium
species and 11 other aerobic actinomycetes by the presence or absence of
Bst
EII recognition sites. Identification of 15 of these 19 isolates was also equivocal by traditional methods. PRA results were reportable within 2 to 5 working days and were as accurate as and faster and less expensive to obtain than those of traditional methods.
Assessment of Three In Vitro Tests and an In Vivo Test for Chloroquine Resistance in
Plasmodium falciparum
Clinical Isolates
Authors: Bickii Jean , Basco Leonardo K. , Ringwald Pascal
Published: December 2019

Three in vitro assays (the isotopic semimicrotest [700 μl per well; 24-well plates], the isotopic microtest [200 μl per well; 96-well plates], and the rapid in vitro test) and the standard in vivo test for chloroquine resistance were compared for 99 clinical isolates of
Plasmodium falciparum
obtained from symptomatic African patients. The 50% inhibitory concentrations determined by the two isotopic tests were similar and were highly correlated (
r
= 0.965;
P
< 0.05), showing a high concordance between the semimicrotest and the microtest. There was a moderate agreement between these two isotopic tests and the in vivo test. Most of the discordant results were probably due to host factors, including reinfections, pharmacokinetic variations, and immunologic response, which are eliminated in in vitro assays. The rapid in vitro test based on the inhibition of chloroquine efflux in the presence of verapamil was poorly concordant with the other tests. Despite some discordant results, isotopic in vitro assays are useful to characterize the phenotypes of individual isolates without the interference of host factors and are complementary to in vivo evaluation of drug efficacy. However, in vitro assays need to be standardized to allow direct comparison of results between different laboratories.
First Report of a Human Isolate of
Erwinia persicinus
Authors: O’Hara Caroline M. , Steigerwalt Arnold G. , Hill Bertha C. , Miller J. Michael , Brenner Don J.
Published: December 2019

Erwinia persicinus
was first described in 1990 after being isolated from a variety of fruits and vegetables, including bananas, cucumbers, and tomatoes. In 1994, it was shown to be the causative agent of necrosis of bean pods. We now report the first human isolate of
E. persicinus
. The strain was isolated from the urine of an 88-year-old woman who presented with a urinary tract infection. By the hydroxyapatite method, DNA from this strain was shown to be 94.5% related at 60°C and 86% related at 75°C to the type strain of
E. persicinus
. The biochemical profile of
E. persicinus
is most similar to those of
Erwinia rhapontici
,
Pantoea agglomerans
, and
Enterobacter
species. It is negative in tests for lysine, arginine, ornithine, dulcitol, and urea. It is motile and positive in tests for
d
-sorbitol and sucrose. It is susceptible to the expanded-spectrum cephalosporins, aminoglycosides, and fluoroquinolones, but it is resistant to ampicillin, ticarcillin, and cefazolin.
Sporadic Emergence of
Klebsiella pneumoniae
Strains Resistant to Cefepime and Cefpirome in Greek Hospitals
Authors: Tzouvelekis L. S. , Tzelepi E. , Prinarakis E. , Gazouli M. , Katrahoura A. , Giakkoupi P. , Paniara O. , Legakis N. J.
Published: December 2019

The sporadic emergence of
Klebsiella pneumoniae
strains resistant to cefepime and cefpirome was observed in Greek hospitals during 1996. Examination of six epidemiologically distinct strains and clones selected in vitro provided indications that resistance is due to the cooperation of decreased outer membrane permeability and hydrolysis of the cephalosporins by SHV-5 β-lactamase, which was produced in large amounts.
Evaluation of Screening and Commercial Methods for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci
Authors: Hussain Zafar , Stoakes Luba , Lannigan Robert , Longo Susan , Nancekivell Barbara
Published: December 2019

The National Committee for Clinical Laboratory Standards recommends 48 h of incubation by the oxacillin salt agar screen (OSAS) method for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the
mecA
gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6
mecA
-positive strains were phenotypically methicillin susceptible. We conclude that most methicillin-resistant CoNS can be detected and the results can be reported after overnight incubation by a combination of methods.
Low Sensitivity of Peripheral Blood Smear for Diagnosis of Subclinical Visceral Leishmaniasis in Human Immunodeficiency Virus Type 1-Infected Patients
Authors: Delgado J. , Pineda J. A. , Macías J. , Regordán C. , Gallardo J. A. , Leal M. , Sanchez-Quijano A. , Lissen E.
Published: December 2019

The peripheral blood smear is an easy method for the diagnosis of symptomatic visceral leishmaniasis (VL) in human immunodeficiency virus type 1 (HIV-1)-infected patients. However, its efficiency in diagnosing subclinical VL remains unknown. In this study,
Leishmania
amastigotes were seen in blood smears from 1 of 13 HIV-1-positive individuals with subclinical VL. This shows that this procedure is not suitable for subclinical-VL diagnosis.
Comparison of ATB Staph, Rapid ATB Staph, Vitek, and E-Test Methods for Detection of Oxacillin Heteroresistance in Staphylococci Possessing
mecA
Authors: Frebourg Noëlle Barbier , Nouet Delphine , Lemée Ludovic , Martin Emmanuelle , Lemeland Jean-François
Published: December 2019

The performance characteristics of the E-test (AB Biodisk, Solna, Sweden), the ATB Staph, the Rapid ATB Staph, and the Vitek GPS-503 card (bioMérieux, La Balme Les Grottes, France) methods for the detection of oxacillin resistance in a collection of staphylococci with a high proportion of troublesome strains were evaluated. Sixty-four
Staphylococcus aureus
strains and 76 coagulase-negative staphylococcal strains were tested. All strains were
mecA
positive and were characterized by the oxacillin agar screen plate test; 75 (53.6%) were found to be heterogeneous by a large-inoculum oxacillin disk diffusion assay, and oxacillin MICs for 89 (63.6%) were ≤32 μg/ml. Three (4.7%)
S. aureus
strains and 25 (32.9%) coagulase-negative strains were classified as susceptible by the E-test, as defined by the National Committee for Clinical Laboratory Standards (NCCLS) oxacillin breakpoint (MIC ≤ 2 μg/ml). The ATB Staph method failed to detect oxacillin resistance in 7 (11%)
S. aureus
isolates and 32 (42.1%) coagulase-negative isolates. The MICs for all but six of these discrepant isolates were ≤16 μg/ml. The Rapid ATB Staph method was tested against
S. aureus
strains only and yielded 15 (23.4%) false-susceptible results for strains for which the MICs were ≤32 μg/ml. The Vitek system was the best-performing system, since it failed to detect oxacillin resistance in only 3 (4.7%)
S. aureus
strains and 15 (19.7%) coagulase-negative strains, the MICs for all of which were ≤2 μg/ml. These data indicate that (i) the performance of the two ATB Staph systems can be limited when the prevalence of borderline-heteroresistant staphylococci is high and (ii) the unreliability of the E-test and the Vitek methods for detecting resistant coagulase-negative strains might be reduced by the potential revision of the oxacillin breakpoint currently recommended by the NCCLS.
Prospective Investigation of Cryptic Outbreaks of
Salmonella agona
Salmonellosis
Authors: Taylor Jeffery P. , Barnett Ben J. , del Rosario Lemuel , Williams Karen , Barth Suzanne S.
Published: December 2019

The number of
Salmonella agona
isolates reported annually in Texas from 1992 through 1994 ranged from 14 to 21. An increase in incidence of
S. agona
infections was noted in the fall of 1995. Pulsed-field gel electrophoresis (PFGE) analysis identified prospectively two possible cryptic outbreaks caused by an indistinguishable strain which was isolated from 18 of 59 patients who were culture positive from March through December 1995. These 18 patients had onset of illness from 20 May through 3 October 1995. Eight individuals resided in the Austin area, eight resided in San Antonio, and two resided in Houston; none had attended a common social gathering or owned common pets. Six patients in San Antonio and one patient from Houston recalled eating food items from the same Mexican food restaurant in San Antonio.
S. agona
organisms with the same PFGE profile were isolated from machacado, an air-dried, raw beef product prepared at the restaurant. The machacado had been shredded in a kitchen blender which was the probable source for cross-contamination of other food items. Five patients in Austin reported eating at a popular Mexican food restaurant in Austin. Improperly prepared machacado also may have been served at the Austin restaurant; however, sufficient quantities of machacado were not available for analysis. PFGE was essential in determining whether the cases constituted outbreaks and was invaluable in guiding the epidemiological investigation.
Direct Antimicrobial Susceptibility Testing of Gram-Negative Bacilli in Blood Cultures by an Electrochemical Method
Authors: Huang Ay Huey , Wu Jiunn Jong , Weng Yu Mei , Ding Hwia Cheng , Chang Tsung Chain
Published: December 2019

Nonfastidious aerobic gram-negative bacilli (GNB) are commonly isolated from blood cultures. The feasibility of using an electrochemical method for direct antimicrobial susceptibility testing of GNB in positive blood cultures was evaluated. An aliquot (10 μl) of 1:10-diluted positive blood cultures containing GNB was inoculated into the Bactometer module well (bioMérieux Vitek, Hazelwood, Mo.) containing 1 ml of Mueller-Hinton broth supplemented with an antibiotic. Susceptibility tests were performed in a breakpoint broth dilution format, with the results being categorized as resistant, intermediate, or susceptible. Seven antibiotics (ampicillin, cephalothin, gentamicin, amikacin, cefamandole, cefotaxime, and ciprofloxacin) were used in this study, with each agent being tested at the two interpretive breakpoint concentrations. The inoculated modules were incubated at 35°C, and the change in impedance in each well was continuously monitored for 24 h by the Bactometer. The MICs of the seven antibiotics for each blood isolate were also determined by the standardized broth microdilution method. Of 146 positive blood cultures (1,022 microorganism-antibiotic combinations) containing GNB tested by the direct method, the rates of very major, major, and minor errors were 0, 1.1, and 2.5%, respectively. The impedance method was simple; no centrifugation, preincubation, or standardization of the inocula was required, and the susceptibility results were normally available within 3 to 6 h after inoculation. The rapid method may allow proper antimicrobial treatment almost 30 to 40 h before the results of the standard methods are available.