bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Protective Efficacy of DNA Vaccines Expressing Secreted Proteins ofMycobacterium tuberculosis
Authors: Kamath Arun T. , Feng Carl G. , Macdonald Murdo , Briscoe Helen , Britton Warwick J.
Published: March 2020

The development of more-effective antituberculosis vaccines would assist in the control of the global problem of infection withMycobacterium tuberculosis. One recently devised vaccination strategy is immunization with DNA plasmids encoding individual microbial genes. Using the genes for theM. tuberculosissecreted proteins MPT64 (23 kDa), Ag85B (30 kDa), and ESAT-6 (6 kDa) as candidate antigens, DNA vaccines were prepared and tested for immunogenicity and protective efficacy in a murine model of aerosolized tuberculosis (TB). Intramuscular immunization with DNA-64 or DNA-85B resulted in the activation of CD4+T cells, which produce gamma interferon (IFN-γ), and high titers of specific immunoglobulin G antibodies. Further, DNA-64 induced major histocompatibility complex class I-restricted CD8+cytotoxic T cells. The addition of a eukaryotic leader sequence tompt64did not significantly increase the T-cell or antibody response. Each of the three DNA vectors stimulated a significant reduction in the level ofM. tuberculosisinfection in the lungs of mice challenged 4 weeks after immunization, but not to the levels resulting after immunization withMycobacterium bovisBCG. The vaccines showed a consistent hierarchy of protection, with the most effective being Ag85B, followed by ESAT-6 and then MPT64. Coimmunization with the three vectors resulted in a greater degree of protection than that induced by any single vector. This protective efficacy was associated with the emergence of IFN-γ-secreting T cells earlier than in infected animals immunized with a control vector. The efficacy of these DNA vaccines suggests that multisubunit vaccination may contribute to future vaccine strategies against TB.
Protein H, an Antiphagocytic Surface Protein inStreptococcus pyogenes
Authors: Kihlberg Britt-Marie , Collin Mattias , Olsén Arne , Björck Lars
Published: March 2020

Surface-associated M protein is a major virulence factor inStreptococcus pyogeneswhich confers bacterial resistance to phagocytosis. However, manyS. pyogenesstrains also express additional structurally related so-called M-like proteins. The strain studied here is of the clinically important M1 serotype and expresses two structurally related surface proteins, the M1 protein and protein H. Mutants were generated that expressed only one or none of these proteins at the bacterial surface. For survival in human blood either protein H or M1 protein was sufficient, whereas the double mutant was rapidly killed. The protein-binding properties of protein H, M1 protein, and the mutants suggest that bacterial binding of immunoglobulin G and factor H or factor H-like protein 1, which are regulatory proteins in the complement system, contribute to the antiphagocytic property.
A Novel Urease-Negative
Helicobacter
Species Associated with Colitis and Typhlitis in IL-10-Deficient Mice
Authors: Fox James G. , Gorelick Peter L. , Kullberg Marika C. , Ge Zhongming , Dewhirst Floyd E. , Ward Jerrold M.
Published: March 2020

A spiral-shaped bacterium with bipolar, single-sheathed flagella was isolated from the intestines of IL-10 (interleukin-10)-deficient (IL-10
−/−
) mice with inflammatory bowel disease. The organism was microaerobic, grew at 37 and 42°C, and was oxidase and catalase positive but urease negative. On the basis of 16S rRNA gene sequence analysis and biochemical and phenotypic criteria, the organism is classified as a novel helicobacter. Cesarean section-rederived IL-10
−/−
mice without helicobacter infection did not have histological evidence of intestinal inflammation. However, helicobacter-free IL-10
−/−
, SCID/NCr, and A/JNCr mice experimentally inoculated with the novel urease-negative
Helicobacter
sp. developed variable degrees of inflammation in the lower intestine, and in immunocompetent mice, the experimental infection was accompanied by a corresponding elevated immunoglobulin G antibody response to the novel
Helicobacter
sp. antigen. These data support other recent studies which demonstrate that multiple
Helicobacter
spp. in both naturally and experimentally infected mice can induce inflammatory bowel disease. The mouse model of helicobacter-associated intestinal inflammation should prove valuable in understanding how specific microbial antigens influence a complex disease process.
Extracellular Cysteine Protease Produced byStreptococcus pyogenesParticipates in the Pathogenesis of Invasive Skin Infection and Dissemination in Mice
Authors: Lukomski Slawomir , Montgomery Charles A. , Rurangirwa Jacqueline , Geske Robert S. , Barrish James P. , Adams Gerald J. , Musser James M.
Published: March 2020

The role of an extracellular cysteine protease encoded by thespeBgene in group AStreptococcus(GAS) skin infection was studied with a mouse model. Mice were injected subcutaneously with a wild-type GAS serotype M3 strain or a cysteine protease-inactivated isogenic derivative grown to stationary phase. The mortality rate of mice injected with the M3speBmutant strain was significantly decreased (P< 0.0008) compared to that of animals injected with the wild-type parental organism. The abscesses formed in animals infected with the cysteine protease mutant strain were significantly smaller (P< 0.0001) than those caused by the wild-type organism and slowly regressed over 3 to 4 weeks. In striking contrast, infection with the wild-type GAS isolate generated necrotic lesions, and in some animals the GAS disseminated widely from the injection site and produced extensive cutaneous damage. All of these animals developed bacteremia and died. GAS dissemination was accompanied by severe tissue and blood vessel necrosis. Cysteine protease expression in the infected tissue was identified by immunogold electron microscopy. These data demonstrate that cysteine protease expression contributes to soft tissue pathology, including necrosis, and is required for efficient systemic dissemination of the organism from the initial site of skin inoculation.
Human Embryonic Gastric Xenografts in Nude Mice: a New Model of
Helicobacter pylori
Infection
Authors: Lozniewski Alain , Muhale Filipe , Hatier Renee , Marais Armelle , Conroy Marie-Christine , Edert Danielle , le Faou Alain , Weber Michele , Duprez Adrien
Published: March 2020

In vitro or animal models have been used to investigate the pathogenesis of
Helicobacter pylori
infection. However, extrapolation to humans of results obtained with these heterologous models remains difficult. We have developed a new model for the study of
H. pylori
infection that uses human entire embryonic stomachs engrafted in nude mice. At 80 days after implantation, 22 of these xenografts, which exhibited a mature gastric epithelium, were inoculated with 10
7
to 10
8
CFU of either
H. pylori
LB1, a freshly isolated
H. pylori
strain (
n
= 12), or
H. pylori
ATCC 49503 (
n
= 10). After 12-week examination,
H. pylori
LB1 persistently colonized the antrum of all inoculated grafts, as assessed by culture (mucus and mucosa), immunohistochemistry (mucosa), and a rapid urease test (mucus).
H. pylori
ATCC 49503, either before or after in vivo passage, permitted only a transient 2-week colonization in one of the five inoculated grafts in both groups. Colonization was always associated with an increase of gastric juice pH. A mild neutrophil infiltration of the gastric mucosa was noted solely in infected grafts. Transmission electron microscopy showed adherence of
H. pylori
organisms to epithelial cell surface. In six animals, intracytoplasmic location of this bacterium was observed in the antrum or the fundus. These results allow us to propose this model as a new ex vivo model for the study of specific
H. pylori
-gastric cell interactions.
Effects of Interleukin-1 Receptor Antagonist Overexpression on Infection byListeria monocytogenes
Authors: Irikura Vera M. , Hirsch Emmet , Hirsh David
Published: March 2020

Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring cytokine whose only known function is the inhibition of interleukin-1 (IL-1). Using a reverse genetic approach in mice, we previously showed that increasing IL-1ra gene dosage leads to reduced survival of a primary listerial infection. In this study, we characterize further the role of endogenously produced IL-1ra and, by inference, IL-1 in murine listeriosis. IL-1ra overexpression inhibits, but does not eliminate, primary immune responses, reducing survival and increasing bacterial loads in the target organs. We demonstrate that IL-1ra functions in the innate immune response to regulate the peak leukocyte levels in the blood, the accumulation of leukocytes at sites of infection, and the activation of macrophages during a primary infection. Reduced macrophage class II major histocompatibility complex expression was observed despite increased gamma interferon (IFN-γ) levels, suggesting that IL-1 activity is essential along with IFN-γ for macrophage activation in vivo. We also show that IL-1ra plays a more limited role during secondary listeriosis, blunting the strength of the delayed-type hypersensitivity response to listerial antigen while not significantly altering cellular immunity to a second infectious challenge. When these results are compared to those for other mutant mice, IL-1ra appears to be unique among the cytokines studied to date in its regulation of leukocyte migration during primary listeriosis.
Characterization of the Gene Encoding a 26-Kilodalton Protein (OMP26) from Nontypeable
Haemophilus influenzae
and Immune Responses to the Recombinant Protein
Authors: El-Adhami Wafa , Kyd Jennelle M. , Bastin David A. , Cripps Allan W.
Published: March 2020

A 26-kDa protein (OMP26) isolated and purified from nontypeable
Haemophilus influenzae
(NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the
H. influenzae
Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.
Vaccination with Cathepsin L Proteinases and with Leucine Aminopeptidase Induces High Levels of Protection against Fascioliasis in Sheep
Authors: Piacenza Lucía , Acosta Daniel , Basmadjian Isabel , Dalton John P. , Carmona Carlos
Published: March 2020

The potential of different parasite proteinases for use as vaccine candidates against fascioliasis in sheep was studied by vaccinating animals with the cathepsin L proteinases CL1 and CL2 and with leucine aminopeptidase (LAP) purified from adult flukes. In the first trial, sheep were immunized with CL1 or CL2 and the mean protection levels obtained were 33 and 34%, respectively. Furthermore, a significant reduction in egg output was observed in sheep vaccinated either with CL1 (71%) or with CL2 (81%). The second trial was performed to determine the protective potential of the two cathepsin L proteinases assayed together, as well as in combination with LAP, and of LAP alone. The combination of CL1 and CL2 induced higher levels of protection (60%) than those produced when these enzymes were administered separately. Those sheep that received the cocktail vaccine including CL1, CL2, and LAP were significantly protected (78%) against metacercarial challenge, but vaccination with LAP alone elicited the highest level of protection (89%). All vaccine preparations induced high immunoglobulin G titers which were boosted after the challenge infection, but no correlations between antibody titers and worm burdens were found. However, the sera of those animals vaccinated with LAP contained LAP-neutralizing antibodies. Reduced liver damage, as assessed by the level of the liver enzyme gamma-glutamyl transferase, was observed in the groups vaccinated with CL1, CL2, and LAP or with LAP alone.
Genetic Control of Experimental Lyme Arthritis in the Absence of Specific Immunity
Authors: Brown Charles R. , Reiner Steven L.
Published: March 2020

Host genetics play an important role in determining resistance or susceptibility to experimental Lyme arthritis. While specific immunity appears to regulate disease resolution, innate immunity appears to regulate disease severity. Intradermal infection withBorrelia burgdorferiyields severe arthritis in C3H/He (C3H) mice but only minimal arthritis in BALB/c mice. Intradermal infection of immunodeficient C3H SCID mice also results in severe arthritis, but arthritis of only moderate severity in BALB/c SCID mice. In the present study, we examined immunodeficient recombinase-activating gene-knockout (RAG-1−/−) (RAG−) mice from resistant C57BL/6 (B6) and DBA/2 (DBA) mouse strains.B. burgdorferi-infected B6 RAG−and DBA RAG−mice had little or no ankle swelling, a low occurrence of inflammatory infiltrates in tibiotarsal joints, and low arthritis severity scores in comparison to RAG+and RAG−BALB/c or C3H mice. Few differences in spirochete DNA levels in ankles of resistant and susceptible RAG−mice were seen. These data suggest that resistance to arthritis development followingB. burgdorferiinfection is not necessarily dependent on an acquired immune response and can occur despite the presence of high spirochete burden. Thus, genes expressed outside the specific immune response can be central regulators of experimental arthritis.
Salmonella typhimuriumEncodes a Putative Iron Transport System within the Centisome 63 Pathogenicity Island
Authors: Zhou Daoguo , Hardt Wolf-Dietrich , Galán Jorge E.
Published: March 2020

Upon entry into the host,Salmonella entericastrains are presumed to encounter an iron-restricted environment. Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment. We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island ofSalmonella typhimurium. The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of theS. typhimuriumchromosome. This locus, designatedsit, consists of four open reading frames which encode polypeptides with extensive homology to theyfeABC iron transport system ofYersinia pestis, as well as other ABC transporters. ThesitAgene encodes a putative periplasmic binding protein,sitBencodes an ATP-binding protein, andsitCandsitDencode two putative permeases (integral membrane proteins). This operon is capable of complementing the growth defect of the enterobactin-deficientEscherichia colistrain SAB11 in iron-restricted minimal medium. Transcription of thesitoperon is repressed under iron-rich growth conditions in afur-dependent manner. Introduction of asitBCDdeletion into wild-typeS. typhimuriumresulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype. These results further support the existence of redundant iron uptake systems inS. enterica.
Segmented Filamentous Bacteria Are Potent Stimuli of a Physiologically Normal State of the Murine Gut Mucosal Immune System
Authors: Talham Gwen L. , Jiang Han-Qing , Bos Nicolaas A. , Cebra John J.
Published: March 2020

Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer’s patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4+T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal,Morganella morganii, to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability ofM. morganiito translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4+, CD45RBlowT cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization withM. morganiiwas able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation ofM. morganiiin the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.
Transcriptional Analysis of the
Staphylococcus aureus
Penicillin Binding Protein 2 Gene
Authors: Pinho Mariana G. , de Lencastre Herminia , Tomasz Alexander
Published: March 2020

Sequencing of the vicinity of the staphylococcal
pbp2
gene and transcriptional analysis by primer extension and promoter fusions were used to show that
pbp2
is part of an operon that also includes a gene with high homology to
prfA
of
Bacillus subtilis
. Two distinct promoters were identified directing transcription of
pbp2
either alone or together with
prfA
. It was recently reported that transposon inactivation of
pbp2
causes a reduction in methicillin resistance, but complementation experiments were not fully successful. We now show that introduction of the intact
pbp2
gene with its two newly identified promoters into the chromosome of the transposon mutant resulted in the full recovery of high-level methicillin resistance.
Expression of
lacZ
from the Promoter of the
Escherichia coli spc
Operon Cloned into Vectors Carrying the W205
trp-lac
Fusion
Authors: Liang Sung-Tzu , Dennis Patrick P. , Bremer Hans
Published: March 2020

The expression of
lacZ
has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of
lacZ
mRNA by hybridization and the amount of β-galactosidase by standard enzymatic assay. Expression was driven by the promoter, P

spc

, of the
spc
ribosomal protein operon. The vectors contained either the standard W205
trp-lac
fusion with the
trp
operon transcription terminator,
trpt
, located in the
lacZ
leader sequence, or a deletion derivative that functionally inactivates
trpt
. In the presence of
trpt
,
lacZ
expression was temperature dependent so that increasing the growth temperature reduced the accumulation of
lacZ
mRNA and β-galactosidase activity. The frequency of transcript termination at
trpt
was estimated to be near zero at 20°C and at about 45% at 37°C. The amount of P

spc

-derived
lacZ
mRNA and the amount of β-galactosidase produced per
lacZ
mRNA varied, depending on the mRNA 5′ leader sequence between P

spc

and
lacZ
. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation. One particular construct without
trpt
did not seem to contain fortuitous transcription or translation signals generated at the fusion junction. In this strain,
lacZ
expression from P

spc

was compared at the enzyme activity and mRNA levels with a previously constructed strain in which
lacZ
was linked to the tandem P1 and P2 promoters of the
rrnB
operon. At any given growth rate, the different activities of β-galactosidase in these two strains were found to reflect the same differences in their amounts of
lacZ
mRNA. Assuming that the promoter-
lacZ
fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of P

spc

and the relative translation efficiency of P

spc

-
lacZ
mRNA at different growth rates. Transcription from the
spc
promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h. The translation frequency of
lacZ
mRNA expressed from P

spc

was unaffected by growth rates.
Genetic and Sequence Analysis of the pTiC58
trb
Locus, Encoding a Mating-Pair Formation System Related to Members of the Type IV Secretion Family
Authors: Li Pei-Li , Everhart Dawn M. , Farrand Stephen K.
Published: March 2020

Conjugal transfer of pTiC58 requires two regions,
tra
which contains the
oriT
and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o’clock position of the plasmid. Using transposon mutagenesis with Tn
3
HoHo1 and a binary transfer system, we delimited this second region, called
trb
, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encoding
traI
, already has been described and is responsible for the synthesis of
Agrobacterium
autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639–4643, 1994). Translation products of the next 11 ORFs showed similarities to those of
trbB
,
-C
,
-D
,
-E
,
-J
,
-K
,
-L
,
-F
,
-G
,
-H
, and
-I
of the
trb
region of the octopine-type Ti plasmid pTi15955 and of the
tra2
core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the
trb
gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the
lacZ
fusions formed by Tn
3
HoHo1, required the
traI
promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the
trb
system of pTiC58 did not allow propagation of the
trb
-specific bacteriophages PRD1, PRR1, and Pf3. The products of several
trb
genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.
The
facC
Gene of
Aspergillus nidulans
Encodes an Acetate-Inducible Carnitine Acetyltransferase
Authors: Stemple Christopher J. , Davis Meryl A. , Hynes Michael J.
Published: March 2020

Mutations in the
facC
gene of
Aspergillus nidulans
result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the
facC
gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in
facC
mutants, while fatty acid-inducible activity was absent in an
acuJ
mutant. Acetate induction of
facC
expression was dependent on the
facB
regulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′
facC
sequences. Carbon catabolite repression of
facC
expression was affected by mutations in the
creA
gene and a CreA fusion protein bound to 5′
facC
sequences. Mutations in the
acuJ
gene led to increased acetate induction of
facC
expression and also of an
amdS-lacZ
reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of
facB
. A model is presented in which
facC
encodes a cytosolic CAT enzyme, while a different CAT enzyme, which is
acuJ
dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments.
Characterization of Mutations That Allow p -Aminobenzoyl-Glutamate Utilization by
Escherichia coli
Authors: Hussein Mouyassar J. , Green Jacalyn M. , Nichols Brian P.
Published: March 2020

An
Escherichia coli
strain deficient in
p
-aminobenzoate synthesis was mutagenized, and derivatives were selected for growth on folic acid. Supplementation was shown to be due to
p
-aminobenzoyl-glutamate present as a breakdown product in commercial folic acid preparations. Two classes of mutations characterized by the minimum concentration of
p
-aminobenzoyl-glutamate that could support growth were obtained. Both classes of mutations were genetically and physically mapped to about 30 min on the
E. coli
chromosome. A cloned wild-type gene from this region,
abgT
(formerly
ydaH
) could confer a similar
p
-aminobenzoyl-glutamate utilization phenotype on the parental strain. Interruption of
abgT
on the plasmid or on the chromosome of the mutant strain resulted in a loss of the phenotype.
abgT
was the third gene in an apparent operon containing
abgA
,
abgB
,
abgT
, and possibly
ogt
and might be regulated by a divergently transcribed LysR-type regulator encoded by
abgR
. Two different single-base-pair mutations that gave rise to the
p
-aminobenzoyl-glutamate utilization phenotype lay in the
abgR-abgA
intercistronic region and appeared to allow the expression of
abgT
. The second class of mutation was due to a tandem duplication of
abgB
and
abgT
fused to
fnr
. The
abgA
and
abgB
gene products were homologous to one another and to a family of aminoacyl aminohydrolases.
p
-Aminobenzoyl-glutamate hydrolysis could be detected in extracts from several of the mutant strains, but intact
abgA
and
abgB
were not essential for
p
-aminobenzoyl-glutamate utilization when
abgT
was supplied in
trans
.
Sister Chromatid Exchange Frequencies in
Escherichia coli
Analyzed by Recombination at the
dif
Resolvase Site
Authors: Steiner Walter W. , Kuempel Peter L.
Published: March 2020

Sister chromatid exchange (SCE) in
Escherichia coli
results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the
dif
resolvase site. Recombination at
dif
is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the
dif
site in a variety of genetic backgrounds and conditions. Recombination at
dif
was increased by known hyperrecombinogenic mutations such as
polA
,
dut
, and
uvrD
. It was also increased by a
fur
mutation, which increased oxidative DNA damage. Recombination at
dif
was eliminated by a
recA
mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in
E. coli
. Interestingly, recombination at
dif
was reduced to approximately half of the wild-type levels by single mutations in either
recB
or
recF
, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.
RecA-Independent Pathways of Lambdoid Prophage Induction in
Escherichia coli
Authors: Rozanov Dmitry V. , D’Ari Richard , Sineoky Sergey P.
Published: March 2020

Two
Escherichia coli
genes, expressed from multicopy plasmids, are shown to cause partial induction of prophage λ in
recA
mutant lysogens. One is
rcsA
, which specifies a positive transcriptional regulator of the
cps
genes, which are involved in capsular polysaccharide synthesis. The other is
dsrA
, which specifies an 85-nucleotide RNA that relieves repression of the
rcsA
gene by histone-like protein H-NS. Genetic contexts known to increase Cps expression also cause RecA-independent λ induction: the
rcsC137
mutation, which causes constitutive Cps expression, and the
lon
and
rcsA3
mutations, which stabilize RcsA. Lambdoid phages 21, φ80, and 434 are also induced by RcsA and DsrA overexpression in
recA
lysogens. Excess λ cI repressor specifically blocks λ induction, suggesting that induction involves repressor inactivation rather than repressor bypass. RcsA-mediated induction requires RcsB, the known effector of the
cps
operon, whereas DsrA-mediated induction is RcsB independent in stationary phase, pointing to the existence of yet another RecA-independent pathway of prophage induction.
Regulation of an Osmoticum-Responsive Gene in
Anabaena
sp. Strain PCC 7120
Authors: Schwartz Steven H. , Black Todd A. , Jäger Karin , Panoff Jean-Michel , Wolk C. Peter
Published: March 2020

Salt-induced genes in the cyanobacterium
Anabaena
sp. strain PCC 7120 were identified by use of a Tn
5
-based transposon bearing
luxAB
as a reporter. The genomic sequence adjacent to one site of insertion of the transposon was identical in part to the sequence of the
lti2
gene, which was previously identified in a differential screen for cold-induced transcripts in
Anabaena variabilis
. The
lti2
-like gene was induced by sucrose and other osmotica and by low temperature, in addition to salt. Regulatory components necessary for the induction of this gene by osmotica were sought by a further round of transposon mutagenesis. One mutant that displayed reduced transcriptional activity of the
lti2
-like gene in response to exposure to osmotica had an insertion in an open reading frame, which was denoted
orrA
, whose predicted product showed sequence similarity to response regulators from two-component regulatory systems. The corresponding mutation was reconstructed and was shown, like the second-site transposon mutation, to result in reduced response to osmotic stress. Induction of the
lux
reporter gene by osmotica was restored by complementation with a genomic fragment containing the entire open reading frame for the presumptive response regulator, whereas a fragment containing a truncated copy of the open reading frame for the response regulator did not complement the mutation.
Effects of Chromosome Underreplication on Cell Division in
Escherichia coli
Authors: Botello Emilia , Nordström Kurt
Published: March 2020

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled
Escherichia coli intR1
strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.
Short-Term Regulation of Nitrogenase Activity by NH
4
+
in
Rhodobacter capsulatus
: Multiple In Vivo Nitrogenase Responses to NH
4
+
Addition
Authors: Yakunin Alexander F. , Hallenbeck Patrick C.
Published: March 2020

The photosynthetic bacterium
Rhodobacter capsulatus
has been shown to carry out nitrogenase “switch-off,” a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a
draT draG
mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a “magnitude” response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH
4
+
.
EnterohemorrhagicEscherichia coli(EHEC) Strains of Serogroup O118 Display Three Distinctive Clonal Groups of EHEC Pathogens
Authors: Wieler Lothar H. , Busse Barbara , Steinrück Hartmut , Beutin Lothar , Weber Albert , Karch Helge , Baljer Georg
Published: March 2020

A recent case report of a child infected with enterohemorrhagicEscherichia coli(EHEC) of serotype O118:H16 in Bavaria, in association with the isolation of a bovine O118 strain on the same farm (A. Weber, H. Klie, H. Richter, P. Gallien, M. Timm, and K. W. Perlberg, Berl. Muench. Tieraerztl. Wochenschr. 110:211–213, 1997), prompted us to investigate the relationship between bovine and human strains of serogroup O118. A total of 29 human O118E. colistrains from Europe (21), Canada (4), and Peru (4) were compared by virulence typing and macrorestriction analysis with 7 bovine O118 EHEC strains isolated in Bavaria. Twenty-five of the human strains were characterized as EHEC. By serotyping and determination of the virulence-associated factors Shiga toxin (stx1 stx2 stx2variants), intimin (eae), and EHEC hemolysin (HlyEHEC), three distinctive groups of O118 human pathogens were identified. Most of the strains belonged to serotype O118:H16, displaying the virulence traits Stx1, intimin, HlyEHEC, and EspP/PssA (group 1). In addition, we identified strains of serotype O118:H12 (Stx2d only; group 2) and of serotype O118:H30 (Stx2 and intimin; group 3). Macrorestriction analysis withBlnI andXbaI revealed that all strains with a single O118 serotype profile (O118:H12, O118:H16, and O118:H30) belonged to one clonal cluster, irrespective of their origin. Group 1 strains clustered in the same clonal group as the bovine O118:H16 strains. Moreover, four pairs of strains of different origins and indistinguishable by all other methods applied were identified as group 1 strains. Our data support the direct transmission of an EHEC O118:H16 strain from a calf to a 2-year-old boy in the above-mentioned case report. Since bovine and human O118:H16 strains represent the same clones, they must be considered zoonotic EHEC pathogens. In contrast, EHEC strains of serotypes O118:H12 and O118:H30 have been isolated only from humans, indicating a reservoir for certain human O118 EHEC strains other than bovines.
Evaluation of Three Rapid Methods for Detection of Methicillin Resistance inStaphylococcus aureus
Authors: Louie L. , Matsumura S. O. , Choi E. , Louie M. , Simor A. E.
Published: March 2020

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistantStaphylococcus aureus(MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistantS. aureus(BORSA;mecA-negative, oxacillin MICs of 2 to 8 μg/ml). The Velogene is a 90-min assay using a chimeric probe to detect themecAgene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR formecAgene detection. A total of 397 clinical isolates ofS. aureuswere tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance inS. aureusin clinical laboratories, especially whenmecAPCR gene detection is unavailable.
Comparison of Serologic Assays and PCR for Diagnosis of Human Herpesvirus 8 Infection
Authors: Spira Thomas J. , Lam Lee , Dollard Sheila C. , Meng Yuan-Xiang , Pau Chou Pong , Black Jodi B. , Burns Darrell , Cooper Brian , Hamid Melissa , Huong Joe , Kite-Powell Kathleen , Pellett Philip E.
Published: March 2020

A variety of assays for the diagnosis human herpesvirus 8 (HHV-8) infection have been reported. We compared several such assays with a panel of 88 specimens from human immunodeficiency virus (HIV)-infected patients with Kaposi's sarcoma (KS) (current-KS patients;
n
= 30), HIV-infected patients who later developed KS (later-KS patients;
n
= 13), HIV-infected patients without KS (no-KS patients;
n
= 25), and healthy blood donors (
n
= 20). PCR assays were also performed with purified peripheral blood mononuclear cells (PBMCs) to confirm positive serologic test results. The order of sensitivity of the serologic assays (most to least) in detecting HHV-8 infection in current-KS patients was the mouse monoclonal antibody-enhanced immunofluorescence assay (MIFA) for lytic antigen (97%), the orfK8.1 peptide enzyme immunoassay (EIA) (87%), the orf65 peptide EIA (87%), MIFA for latent antigen (83%), the Advanced Biotechnologies, Inc., EIA (80%), and the orf65 immunoblot assay (80%). Combination of the results of the two peptide EIAs (combined peptide EIAs) increased the sensitivity to 93%. For detection of infection in later-KS patients, the MIFA for lytic antigen (100%), the orfK8.1 peptide EIA (85%), and combined peptide EIAs (92%) were the most sensitive. Smaller percentages of no-KS patients were found to be positive (16 to 56%). Most positive specimens from the current-KS and later-KS groups were positive by multiple assays, while positive specimens from the no-KS group tended to be positive only by a single assay. PCR with PBMCs for portions of the HHV-8 orf65 and gB genes were positive for less than half of current-KS and later-KS patients and even fewer of the no-KS patients. The concordance between serologic assays was high. We propose screening by the combined peptide EIAs. For specimens that test weakly positive, we recommend that MIFA for lytic antigen be done. A positive result with a titer of ≥1:40 would be called HHV-8 positive. A negative or low titer would be called HHV-8 negative. If a population has a high percentage of persons who test positive by the combined peptide EIAs, then a MIFA could be performed with the negative specimens to determine if any positive specimens are being missed. Alternatively, if a population has a low percentage that test positive, then a MIFA could be performed with a subset of the negative specimens for the same reason. As described above, only a titer of ≥1:40 would be considered HHV-8 positive.
Evaluation of IS200-PCR and Comparison with Other Molecular Markers To TraceSalmonella entericasubsp.entericaSerotype Typhimurium Bovine Isolates from Farm to Meat
Authors: Millemann Yves , Gaubert Stéphane , Remy Dominique , Colmin Catherine
Published: March 2020

A procedure that uses an original molecular marker (IS200-PCR) and that is based on the amplification of DNA with outward-facing primers complementary to each end of IS200has been evaluated with a collection of 85Salmonella entericasubsp.entericaserotype Typhimurium isolates. These strains were isolated from a group of 10 cows at different stages: during transportation between the farm and the slaughterhouse, on the slaughter line, from the environment, and from the final product (ground beef). The 85 isolates were characterized by their antibiotic resistance patterns and were compared by IS200-PCR and by use of four other genotypic markers. Those markers included restriction profiles for 16S and 23S rRNA (ribotypes) and amplification profiles obtained by different approaches: random amplified polymorphic DNA analysis, enterobacterial repetitive intergenic consensus PCR, and PCR ribotyping. The results of the IS200-PCR were in accordance with those of other molecular typing methods for this collection of isolates. Five different genotypes were found, which made it possible to refine the hypotheses on transmission obtained from phenotypic results. The genotyping results indicated the massive contamination of the whole group of animals and of the environment by one clonal strain originally recovered from one cow that excreted the strain. On the other hand, a few animals and their environment appeared to be simultaneously contaminated with genetically different strains.
A Comparison of Seven Tests for Serological Diagnosis of Tuberculosis
Authors: Pottumarthy Sudha , Wells Virginia C. , Morris Arthur J.
Published: March 2020

Seven serological tests, two immunochromatographic tests, ICT Tuberculosis and RAPID TEST TB, and five enzyme-linked immunosorbent assays, TUBERCULOSIS IgA EIA, PATHOZYME-TB complex, PATHOZYME-MYCO IgG, PATHOZYME-MYCO IgA, and PATHOZYME-MYCO IgM, were evaluated simultaneously with 298 serum samples from three groups of individuals: 44 patients with active tuberculosis, 204 controls who had undergone the Mantoux test (89 Mantoux test-positive and 115 Mantoux test-negative controls), and 50 anonymous controls. The sensitivities of the tests with sera from patients with active tuberculosis were poor to modest, ranging from 16 to 57%. All the tests performed equally with sera from subgroups of those with active tuberculosis, those with pulmonary (33 patients) versus extrapulmonary (11 patients) disease, and those who were smear positive (24 patients) versus smear negative (12 patients) (
P
> 0.05). The specificities of the tests ranged from 80 to 97% with sera from the Mantoux test controls and 62 to 100% with sera from the anonymous controls. The TUBERCULOSIS IgA EIA had the highest sensitivity (57%) with sera from patients with active tuberculosis, with a high specificity of 93% with sera from the Mantoux test controls, but a very poor specificity of 62% with sera from the anonymous controls. Overall, ICT Tuberculosis followed by PATHOZYME-MYCO IgG had the best performance characteristics, with sensitivities of 41 and 55%, respectively, with sera from patients with active tuberculosis and specificities of 96 and 89%, respectively, with sera from the Mantoux test controls and 88 and 90%, respectively, with sera from the anonymous controls. By combining all the test results, a maximum sensitivity of 84% was obtained, with reciprocal drops in specificities to 55 and 42% for the Mantoux test controls and anonymous controls, respectively. The best combination was that of ICT Tuberculosis and PATHOZYME-MYCO IgG, with a sensitivity of 66% and a specificity of 86% for the Mantoux test controls and a sensitivity and specificity of 78% for the anonymous controls. While a negative result by any one of these tests would be useful in helping to exclude disease in a population with a low prevalence of tuberculosis, a positive result may aid in clinical decision making when applied to symptomatic patients being evaluated for active tuberculosis.
A Survey of Stool Culturing Practices for
Vibrio
Species at Clinical Laboratories in Gulf Coast States
Authors: Marano Nina N. , Daniels Nicholas A. , Easton Alyssa N. , McShan Andre , Ray Beverly , Wells Joy G. , Griffin Patricia M. , Angulo Frederick J.
Published: March 2020

Non-cholera
Vibrio
infections are an important public health problem. Non-cholera
Vibrio
species usually cause sporadic infections, often in coastal states, and have also caused several recent nationwide outbreaks of gastroenteritis in the United States. We report a survey of laboratory stool culturing practices for
Vibrio
among randomly selected clinical laboratories in Gulf Coast states (Alabama, Florida, Louisiana, Mississippi, and Texas). Interviews conducted with the microbiology supervisors of 201 clinical laboratories found that 164 (82%) received stool specimens for culture. Of these, 102 (62%) of 164 processed stool specimens on site, and 20 (20%) of these 102 laboratories cultured all stool specimens for
Vibrio
, indicating that at least 34,463 (22%) of 152,797 stool specimens were cultured for
Vibrio
. This survey suggests that despite an increased incidence of non-cholera
Vibrio
infections in Gulf Coast states, a low percentage of clinical laboratories routinely screen all stool specimens, and fewer than 25% of stool specimens collected are routinely screened for non-cholera
Vibrio
.
Extraction-Free, Filter-Based Template Preparation for Rapid and Sensitive PCR Detection of Pathogenic Parasitic Protozoa
Authors: Orlandi Palmer A. , Lampel Keith A.
Published: March 2020

Within the last several years, the protozoan parasites
Cyclospora cayetanensis
,
Cryptosporidium parvum
, and microsporidia have become recognized as important, rapidly emerging human pathogens in immunocompromised and immunocompetent individuals. Since the early 1990s, many of the reported outbreaks of enteric illness caused by these microorganisms have been attributed to food- and water-borne contamination. Many inherent obstacles affect the success of current surveillance and detection methods used to monitor and control levels of contamination by these pathogens. Unlike methods that incorporate preenrichment for easier and unambiguous identification of bacterial pathogens, similar methods for the detection of parasitic protozoa either are not currently available or cannot be performed in a timely manner. We have developed an extraction-free, filter-based protocol to prepare DNA templates for use in PCR to identify
C. cayetanensis
and
C. parvum
oocysts and microsporidia spores. This method requires only minimal preparation to partially purify and concentrate isolates prior to filter application. DNA template preparation is rapid, efficient, and reproducible. As few as 3 to 10 parasites could be detected by PCR from direct application to the filters. In studies, as few 10 to 50
Encephalitozoon intestinalis
spores could be detected when seeded in a 100-μl stool sample and 10 to 30
C. cayetanensis
oocysts could be detected per 100 g of fresh raspberries. This protocol can easily be adapted to detect parasites from a wide variety of food, clinical, and environmental samples and can be used in multiplex PCR applications.