bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Evolution of the Pathogenicity Island of Enterotoxigenic
Bacteroides fragilis
Strains
Authors: Franco Augusto A. , Cheng Rodney K. , Chung Gyung-Tae , Wu Shaoguang , Oh Hee-Bok , Sears Cynthia L. 
Published: December 2019

ABSTRACT

Enterotoxigenic
Bacteroides fragilis
(ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic
B. fragilis
(NTBF) strains, we found that
bft
and a second metalloprotease gene (
mpII
) are contained in an ∼6-kb pathogenicity island (termed
B. fragilis
pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (
bfmA
and
bfmB
) organized in one operon and a putative origin of transfer (
oriT
) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (
bfmC
) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from
Escherichia coli
. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that
bfmB
and
bfmC
are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the
bfmB
and
bfmC
stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the
B. fragilis
chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.
Bacterial Phylogenetic Clusters Revealed by Genome Structure
Authors: Liu Shu-Lin , Schryvers Anthony B. , Sanderson Kenneth E. , Johnston Randal N. 
Published: December 2019

ABSTRACT

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-
Ceu
I, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera:
Salmonella
and
Pasteurella
. Among
Salmonella
spp., I-
Ceu
I mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the
Salmonella
spp. In marked contrast, the
Pasteurella
strains have very different genome structures among and even within individual species. The divergence of
Pasteurella
was also reflected in 16S rDNA sequences and far exceeded that seen between
Escherichia
and
Salmonella
. Based on this diversity, the
Pasteurella haemolytica
strains we analyzed could be divided into 14 phylogenetic groups and the
Pasteurella multocida
strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for
Salmonella
and
Escherichia coli
were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of
P. multocida
and
P. haemolytica
to be divided into different species, genera, or even higher ranks. On the other hand, strains of
Pasteurella ureae
and
Pasteurella pneumotropica
are very similar to those of
P. multocida
in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-
Ceu
I-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.
Anaerobic Growth of
Paracoccus denitrificans
Requires Cobalamin: Characterization of
cobK
and
cobJ
Genes
Authors: Shearer Neil , Hinsley Andrew P. , Van Spanning Rob J. M. , Spiro Stephen 
Published: December 2019

ABSTRACT

A pleiotropic mutant of
Paracoccus denitrificans
, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B
12
) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to
cobK
of
Pseudomonas denitrificans
, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with
cobK
is a gene homologous to
cobJ
of
Pseudomonas denitrificans
, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type
P. denitrificans
(which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the
cobK
and
cobJ
genes with their orthologues suggests that
P. denitrificans
uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions,
P. denitrificans
appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that
P. denitrificans
expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.
Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis in
Streptomyces coelicolor
A3(2) as a DNA-Binding Protein
Authors: Arias Paloma , Fernández-Moreno Miguel A. , Malpartida Francisco 
Published: December 2019

ABSTRACT

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the
act
cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type
act
II-ORF4 gene but not with the
act
II-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from
Escherichia coli
cultures; both showed specific DNA-binding activity for the
act
VI-ORF1–ORFA and
act
III-
act
I intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the −35 regions of the corresponding promoters, showing the consensus sequence 5′-TCGAG-3′. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the
act
genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the
act
biosynthetic genes.
Characterization of Activity and Expression of Isocitrate Lyase in
Mycobacterium avium
and
Mycobacterium tuberculosis
Authors: Höner Zu Bentrup Kerstin , Miczak Andras , Swenson Dana L. , Russell David G. 
Published: December 2019

ABSTRACT

Analysis by two-dimensional gel electrophoresis revealed that
Mycobacterium avium
expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the
M. tuberculosis
database (Sanger) with similarity to the enzyme isocitrate lyase of both
Corynebacterium glutamicum
and
Rhodococcus fascians
. Only marginal similarity was observed between this open reading frame (ORF) (termed
icl
) and a second distinct ORF (named
aceA
) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both
M. avium
and
M. tuberculosis
the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.
Physiological Characterization of SusG, an Outer Membrane Protein Essential for Starch Utilization by
Bacteroides thetaiotaomicron
Authors: Shipman Joseph A. , Cho Kyu Hong , Siegel Hilary A. , Salyers Abigail A. 
Published: December 2019

ABSTRACT

Results from previous studies had suggested that
Bacteroides thetaiotaomicron
utilizes starch by binding the polysaccharide to the bacterial surface and subsequently degrading the polymer by using cell-associated enzymes. Most of the starch-degrading activity was localized to the periplasm, but a portion appeared to be membrane associated. This raised the possibility that some breakdown might occur in the outer membrane prior to exposure of the polysaccharide to the periplasmic polysaccharide-degrading enzymes. In this study, we show that SusG, an outer membrane protein which has been shown genetically to be essential for starch utilization, has enzymatic activity. Results of protease accessibility experiments support the hypothesis that SusG is exposed on the cell surface. Results of [
14
C]starch binding assays, however, show that SusG plays a negligible role in binding of starch to the cell surface. Consistent with this, SusG has a relatively high

K
m

for starch and by itself is not sufficient to allow cells to grow on starch or to bind starch. Hence, the main role of SusG is to hydrolyze starch, but the binding of starch to the cell surface is evidently mediated by other proteins presumably interacting with SusG.
Cloning and Analysis of the Capsid Morphogenesis Genes of
Pseudomonas aeruginosa
Bacteriophage D3: Another Example of Protein Chain Mail?
Authors: Gilakjan Zeynab A. , Kropinski Andrew M. 
Published: December 2019

ABSTRACT

The terminal DNA restriction fragments (
Pst
I-D and -B) of
Pseudomonas aeruginosa
bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation (ρ = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor. This is identical to the situation observed with coliphage HK97.
Novel Organization of Genes Involved in Prophage Excision Identified in the Temperate Lactococcal Bacteriophage TP901-1
Authors: Breüner Anne , Brøndsted Lone , Hammer Karin 
Published: December 2019

ABSTRACT

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (
orf7
) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1
attP
site which are integrated into
attB
on the chromosome of
Lactococcus lactis
. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the
upp
gene (encoding uracil phosphoribosyltransferase) was designed, since
upp
mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10
−5
per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.
Negative Regulation of the Gene for Fe-Containing Superoxide Dismutase by an Ni-Responsive Factor in
Streptomyces coelicolor
Authors: Chung Hye-Jung , Choi Jae-Hyun , Kim Eun-Ja , Cho You-Hee , Roe Jung-Hye 
Published: December 2019

ABSTRACT

In
Streptomyces coelicolor
, transcription of the
sodF
genes, encoding Fe-containing superoxide dismutases, is negatively regulated by nickel. Gel mobility shift assays with
sodF1
promoter fragments and cell extracts from the A3(2) strain indicate the presence of a nickel-responsive DNA-binding protein, most likely a transcriptional repressor. The boundary for the Ni-responsive
cis
-acting region was identified both in vitro and vivo. Ni does not regulate the level of the putative repressor but only the binding competence of this protein.
Genome-Wide Transcriptional Analysis of Aerobic and Anaerobic Chemostat Cultures of
Saccharomyces cerevisiae
Authors: ter Linde J. J. M. , Liang H. , Davis R. W. , Steensma H. Y. , van Dijken J. P. , Pronk J. T. 
Published: December 2019

ABSTRACT

The yeast
Saccharomyces cerevisiae
is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen. Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays. This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate. Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions. Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions.
Functional Domains Present in the Mycobacterial Hemagglutinin, HBHA
Authors: Delogu Giovanni , Brennan Michael J. 
Published: December 2019

ABSTRACTIdentification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an α-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.
Genetic Transfer of Large DNA Inserts to Designated Loci of the
Bacillus subtilis
168 Genome
Authors: Itaya Mitsuhiro 
Published: December 2019

ABSTRACT

It was found that contiguous DNA segments of up to 50 kb can be transferred between
Bacillus subtilis
genomes when a sufficient length of the flanking genomic region is provided for homologous recombination, although the efficiency of transfer was reduced as the insert size increased. Inserts were translocated to different loci, where appropriate integration sites were created.
The HPr(Ser) Kinase of
Streptococcus salivarius
: Purification, Properties, and Cloning of the
hprK
Gene
Authors: Brochu Denis , Vadeboncoeur Christian 
Published: December 2019

ABSTRACT

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of
Streptococcus salivarius
ATCC 25975 was purified, and the encoding gene (
hprK
) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the
S. salivarius
enzyme showed 45% identity with the
Bacillus subtilis
enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted
hprK
gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg
2+
for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from
S. salivarius
is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its

K
m

s for HPr and ATP are 31 μM and 1 mM, respectively.
Neisseria gonorrhoeae
PilA Is an FtsY Homolog
Authors: Arvidson Cindy Grove , Powers Ted , Walter Peter , So Magdalene 
Published: December 2019

ABSTRACT

The
pilA
gene of
Neisseria gonorrhoeae
was initially identified in a screen for transcriptional regulators of
pilE
, the expression locus for pilin, the major structural component of gonococcal pili. The predicted protein sequence for PilA has significant homology to two GTPases of the mammalian signal recognition particle (SRP), SRP54 and SRα. Homologs of SRP54 and SRα were subsequently identified in bacteria (Ffh and FtsY, respectively) and appear to form an SRP-like apparatus in prokaryotes. Of the two proteins, PilA is the most similar to FtsY (47% identical and 67% similar at the amino acid level). Like FtsY, PilA is essential for viability and hydrolyzes GTP. The similarities between PilA and the bacterial FtsY led us to ask whether PilA might function as the gonococcal FtsY. In this work, we show that overproduction of PilA in
Escherichia coli
leads to an accumulation of pre-β-lactamase, similar to previous observations with other bacterial SRP components. Low-level expression of
pilA
in an
ftsY
conditional mutant can complement the
ftsY
mutation and restore normal growth to this strain under nonpermissive conditions. In addition, purified PilA can replace FtsY in an in vitro translocation assay using purified
E. coli
SRP components. A PilA mutant that is severely affected in its GTPase activity cannot replace FtsY in vivo or in vitro. However, overexpression of the GTPase mutant leads to the accumulation of pre-β-lactamase, suggesting that the mutant protein may interact with the SRP apparatus to affect protein maturation. Taken together, these results show that the gonococcal PilA is an FtsY homolog and that the GTPase activity is necessary for its function.
Quorum Sensing in
Burkholderia cepacia
: Identification of the LuxRI Homologs CepRI
Authors: Lewenza Shawn , Conway Barbara , Greenberg E. P. , Sokol Pamela A. 
Published: December 2019

ABSTRACT

Burkholderia cepacia
has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible
N
-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of
B. cepacia
K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a
luxR
homolog that was designated
cepR
. The flanking DNA region was used to clone the wild-type copy of
cepR
. Sequence analysis revealed the presence of
cepI
, a
luxI
homolog, located 727 bp upstream and divergently transcribed from
cepR
. A
lux
box-like sequence was identified upstream of
cepI
. CepR was 36% identical to
Pseudomonas aeruginosa
RhlR and 67% identical to SolR of
Ralstonia solanacearum
. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a
cepR
mutation restored parental levels of ornibactin and protease but not lipase. An
N
-acylhomoserine lactone was purified from culture fluids and identified as
N
-octanoylhomoserine lactone. K56-I2, a
cepI
mutant, was created and shown not to produce
N
-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for
cepIR
in the regulation of extracellular virulence factor production by
B. cepacia.