bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Binding of
Escherichia coli
O157:H7 to Alfalfa, Human Epithelial Cells, and Plastic Is Mediated by a Variety of Surface Structures
Authors: Torres Alfredo G. , Jeter Cecelia , Langley William , Matthysse Ann G.
Published: December 2005

Escherichia coli
O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several
E. coli
O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (
cah
,
aidA1
, and
ompA
) and mediators of hyperadherence (
tdcA
,
yidE
,
waaI
, and
cadA
) and those associated with fimbria formation (
csgA
,
csgD
, and
lpfD2
). The introduction of some of these genes (
cah
,
aidA1
, and
csg
loci) into an
E. coli
K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of
E. coli
O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the
ompA
gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a
tdcA ompA
double mutant. In contrast, the
E. coli
O157:H7
ompA
and
tdcA ompA
mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in
E. coli
O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was
ompA
.
Compatible Solutes of the HyperthermophilePalaeococcus ferrophilus: Osmoadaptation and Thermoadaptation in the OrderThermococcales
Authors: Neves Clélia , da Costa Milton S. , Santos Helena
Published: December 2005

The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeonPalaeococcus ferrophilus. The genusPalaeococcusrepresents a deep-branching lineage of the orderThermococcales, which diverged beforeThermococcusandPyrococcus. Palaeococcus ferrophilusaccumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the generaPyrococcusandThermococcus,Palaeococcus ferrophilusdid not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate inPalaeococcus ferrophilusandThermococcus litoraliswere identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). ThempgSandmpgPgenes ofPalaeococcus ferrophiluswere expressed inEscherichia coliand the proteins were characterized. MPGS had maximal activity at 90°C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90°C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83°C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of theThermococcalesis presented.
Relatedness of
Listeria monocytogenes
Isolates Recovered from Selected Ready-To-Eat Foods and Listeriosis Patients in the United States
Authors: Gilbreth Stefanie Evans , Call Jeff E. , Wallace F. Morgan , Scott Virginia N. , Chen Yuhuan , Luchansky John B.
Published: December 2005

Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of
Listeria monocytogenes
, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, ≥66%), 139 AscI pulsotypes (levels of relatedness, ≥25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the
L. monocytogenes
subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.
Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation
Authors: Newcombe David A. , Schuerger Andrew C. , Benardini James N. , Dickinson Danielle , Tanner Roger , Venkateswaran Kasthuri
Published: December 2005

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three
Bacillus
spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m
−2
of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of
Bacillus
species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain,
Bacillus subtilis
168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the
Bacillus
species tested, spores of a
Bacillus pumilus
strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific;
B. pumilus
SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like
B. pumilus
SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.
Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of
Mycobacterium tuberculosis
?
Authors: Gibson Andrea , Brown Timothy , Baker Lucy , Drobniewski Francis
Published: December 2005

The phylogeny and evolution of the bacterium
Mycobacterium tuberculosis
is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469
M. tuberculosis
and 49
Mycobacterium bovis
isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the
M. tuberculosis
complex. This population was characterized by previously identified silent single-nucleotide polymorphisms (sSNPs) or by a macroarray based on these sSNPs that was developed in this study. MIRU-VNTR phylogenetic codes capable of differentiating between phylogenetic lineages were identified. Overall, there was 90.9% concordance between the lineages of isolates as defined by the MIRU-VNTR and sSNP analyses. The MIRU-VNTR phylogenetic code was unique to
M. bovis
and was not observed in any
M. tuberculosis
isolates. The codes were able to differentiate between different
M. tuberculosis
strain families such as Beijing, Delhi, and East African-Indian. Discrepant isolates with similar but not identical MIRU-VNTR codes often displayed a stepwise trend suggestive of bidirectional evolution. A lineage-specific panel of MIRU-VNTR can be used to subdivide each lineage for epidemiological purposes. MIRU-VNTR is a valuable tool for phylogenetic studies and could define an evolutionarily uncharacterized population of
M. tuberculosis
complex organisms.
Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of
Salmonella enterica
Serovar Typhimurium
Authors: Doran Geraldine , Morris Dearbhaile , O'Hare Colette , DeLappe Niall , Bradshaw Bernard , Corbett-Feeney Geraldine , Cormican Martin
Published: December 2005

Salmonella enterica
serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.
Multiple Reductive-Dehalogenase-Homologous Genes Are Simultaneously Transcribed during Dechlorination by
Dehalococcoides-
Containing Cultures
Authors: Waller Alison S. , Krajmalnik-Brown Rosa , Löffler Frank E. , Edwards Elizabeth A.
Published: December 2005

Degenerate primers were used to amplify 14 distinct reductive-dehalogenase-homologous (RDH) genes from the
Dehalococcoides
-containing mixed culture KB1. Most of the corresponding predicted proteins were highly similar (97 to >99% amino acid identity) to previously reported
Dehalococcoides
reductive dehalogenases. To examine the differential transcription of these RDH genes, KB1 was split into five subcultures amended with either trichloroethene,
cis-
1,2-dichloroethene, vinyl chloride, 1,2-dichlorethane, or no chlorinated electron acceptor. Total RNA was extracted following the onset of reductive dechlorination, and RDH transcripts were reverse transcribed and amplified using degenerate primers. The results indicate that the transcription of RDH genes requires the presence of a chlorinated electron acceptor, and for all treatments, multiple RDH genes were simultaneously transcribed, with transcripts of two of the genes being present under all four electron-accepting conditions. Two of the transcribed sequences were highly similar to reported vinyl chloride reductase genes, namely,
vcrA
from
Dehalococcoides
sp. strain VS and
bvcA
from
Dehalococcoides
sp. strain BAV1. These findings suggest that multiple RDH genes are induced by a single chlorinated substrate and that multiple reductive dehalogenases contribute to chloroethene degradation in KB1.
A Broad-Host-Range, Generalized Transducing Phage (SN-T) Acquires 16S rRNA Genes from Different Genera of Bacteria
Authors: Beumer Amy , Robinson Jayne B.
Published: December 2005

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera:
Sphaerotilus natans
, the host from which SN-T was originally isolated, and
Pseudomonas aeruginosa
. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10
−9
transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.
Detection of Genetic Markers of Fecal Indicator Bacteria in Lake Michigan and Determination of Their Relationship to
Escherichia coli
Densities Using Standard Microbiological Methods
Authors: Bower Patricia A. , Scopel Caitlin O. , Jensen Erika T. , Depas Morgan M. , McLellan Sandra L.
Published: December 2005

Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for
Bacteroides
and
Escherichia coli
. Initial experiments with sewage treatment plant influent demonstrated that total
Bacteroides
spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than
E. coli
and human-specific
Bacteroides
spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable
E. coli
for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan.
Bacteroides
spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml
E. coli
. Human-specific
Bacteroides
spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific
Bacteroides
spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers.
Bacteroides
spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of
E. coli
. Human-specific
Bacteroides
spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but
E. coli
levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.
Listeria monocytogenes
PerR Mutants Display a Small-Colony Phenotype, Increased Sensitivity to Hydrogen Peroxide, and Significantly Reduced Murine Virulence
Authors: Rea Rosemarie , Hill Colin , Gahan Cormac G. M.
Published: December 2005

Deletion of
perR
in
Listeria monocytogenes
results in a small-colony phenotype (Δ
perR
sm
) that is slow growing and exhibits increased sensitivity to H
2
O
2
. At a relatively high frequency, large-colony variants (Δ
perR
lg
) arise, which are more resistant to H
2
O
2
than the wild-type and ultimately dominate the culture. Transcriptional analysis revealed that the
kat
gene (catalase) is up-regulated in both types of mutants and that the highest level is apparent in Δ
perR
sm
mutants, demonstrating PerR regulation of this gene. Overexpression of the catalase gene in the wild-type background resulted in a slower-growing strain with a smaller colony size similar to that of Δ
perR
sm
. By combining a bioinformatic approach with experimental evidence, other PerR-regulated genes were identified, including
fur
, lmo0641,
fri
, lmo1604,
hemA
, and
trxB
. The transcriptional profile of these genes in both mutant backgrounds was similar to that of catalase in that a higher level of expression was observed in Δ
perR
sm
than in the wild type or Δ
perR
lg
. Murine studies revealed that the virulence potential of the Δ
perR
sm
mutant is substantially reduced compared to that of the wild-type and Δ
perR
lg
strains. Collectively, the data demonstrate that the Δ
perR
sm
mutant represents the true phenotype associated with the absence of PerR, which is linked to overexpression of regulated genes that negatively affect bacterial homeostasis both in vitro and in vivo. A subsequent secondary mutation occurred at a high frequency, which resulted in phenotypic reversion to a large-colony phenotype with increased fitness that may have obstructed the analysis of the role of PerR in the physiology of the bacterial cell.
Influence of Inorganic Nitrogen Management Regime on the Diversity of Nitrite-Oxidizing Bacteria in Agricultural Grassland Soils
Authors: Freitag Thomas E. , Chang Lisa , Clegg Christopher D. , Prosser James I.
Published: December 2005

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized
Nitrospira-
like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of
Nitrospira-
like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized
Nitrospira
species. Clone libraries of
Nitrobacter
-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate
Nitrobacter
sp. strain PJN1. However, the diversity of
Nitrobacter
communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.
Genetic Variation among
Staphylococcus aureus
Strains from Norwegian Bulk Milk
Authors: Jørgensen H. J. , Mørk T. , Caugant D. A. , Kearns A. , Rørvik L. M.
Published: December 2005

Strains of
Staphylococcus aureus
obtained from bovine (
n
= 117) and caprine (
n
= 114) bulk milk were characterized and compared with
S. aureus
strains from raw-milk products (
n
= 27), bovine mastitis specimens (
n
= 9), and human blood cultures (
n
= 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized
S. aureus
. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that
S. aureus
from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolate
s
within PFGE clusters generally belonged to the same clonal complex.
Complete Sequences of Four Plasmids ofLactococcus lactissubsp.cremorisSK11 Reveal Extensive Adaptation to the Dairy Environment
Authors: Siezen Roland J. , Renckens Bernadet , van Swam Iris , Peters Sander , van Kranenburg Richard , Kleerebezem Michiel , de Vos Willem M.
Published: December 2005

Lactococcus lactisstrains are known to carry plasmids encoding industrially important traits.L. lactissubsp.cremorisSK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologousrepB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside onL. lactisSK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP,pepO,pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while thepabAandpabBgenes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependentd-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before onL. lactisplasmids. Moreover, several genes were identified for the first time inL. lactis, possibly reflecting horizontal gene transfer.
Detection and Cultivation of Soil Verrucomicrobia
Authors: Sangwan Parveen , Kovac Suzana , Davis Kathryn E. R. , Sait Michelle , Janssen Peter H.
Published: December 2005

Only one isolate each of the class “
Spartobacteria
” (subdivision 2 of the phylum
Verrucomicrobia
) and of subdivision 3 of
Verrucomicrobia
have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class “
Spartobacteria
” and were related to “
Chthoniobacter flavus
.” Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class “
Spartobacteria
” were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class “
Spartobacteria
” were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class “
Spartobacteria
” was inhibited by other culturable bacteria.
Enzymatic Ability of
Bifidobacterium animalis
subsp.
lactis
To Hydrolyze Milk Proteins: Identification and Characterization of Endopeptidase O
Authors: Janer C. , Arigoni F. , Lee B. H. , Peláez C. , Requena T.
Published: December 2005

The proteolytic system of
Bifidobacterium animalis
subsp.
lactis
was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in
Bifidobacterium
spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of
B. animalis
subsp.
lactis
were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in
B. animalis
subsp.
lactis
. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of
B. animalis
subsp.
lactis
was examined in more detail, and the gene encoding an endopeptidase O in
B. animalis
subsp.
lactis
was cloned and overexpressed in
Escherichia coli
. The deduced amino acid sequence for
B. animalis
subsp.
lactis
PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from
Bifidobacterium longum
. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of
B. animalis
subsp.
lactis
PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide α
s1
-casein(f1-23). The predominant peptide bond cleaved by
B. animalis
subsp.
lactis
PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.
Gibberella xylarioides
Sensu Lato from
Coffea canephora
: a New Mating Population in the
Gibberella fujikuroi
Species Complex
Authors: Lepoint Pascale C. E. , Munaut Françoise T. J. , Maraite Henri M. M.
Published: December 2005

Gibberella xylarioides
Heim & Saccas (presumed anamorph,
Fusarium xylarioides
Steyaert) is the causal agent of coffee wilt disease, an economically important tracheomycosis in Africa. In vitro crosses carried out with Congolese, Ugandan, and Tanzanian single-ascospore/conidial isolates originating from diseased
Coffea canephora
/
excelsa
demonstrated a heterothallic mating system, controlled by a single locus with two alleles,
MAT-1
and
MAT-2
. Compatible isolates produced fertile perithecia within 2 to 8 weeks after mating. Mating type (
MAT
) was characterized by PCR with primer pairs previously developed for the
Gibberella fujikuroi
species complex (GFC) and for
Fusarium oxysporum
. All strains analyzed were morphologically identical and corresponded to Booth's description of the “female”
F. xylarioides
strain. Based on crossing results and
MAT-2
/translation elongation 1-α (
tef
) sequence data,
G. xylarioides
, as currently understood, is demonstrated to encompass at least three “groups”:
G. xylarioides
sensu strictu Ia, defined hitherto by two “historical” West African strains originating from the severe 1930s to 1950s epidemic (CBS 25852 and CBS 74979);
G. xylarioides
sensu strictu Ib, defined by two “historical” Central African lowland strains (DSMZ 62457 and ATCC 15664); and
G. xylarioides
sensu lato II, containing Congolese, Ugandan, and Tanzanian
C. canephora
/
excelsa
isolates. Infertility of crosses between the coffee wilt pathogen and known GFC mating populations demonstrates that
G. xylarioides
sensu lato constitutes a new biological species within the
G. fujikuroi
complex. MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 are proposed as
G. xylarioides
sensu lato II
MAT-1
/
MAT-2
reference mating type tester strains.
Multilocus Sequence Type System for the Plant Pathogen
Xylella fastidiosa
and Relative Contributions of Recombination and Point Mutation to Clonal Diversity
Authors: Scally Mark , Schuenzel Erin L. , Stouthamer Richard , Nunney Leonard
Published: December 2005

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen
Xylella fastidiosa
using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the
X. fastidiosa
subsp.
fastidiosa
and
X. fastidiosa
subsp.
sandyi
clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of
X. fastidiosa
subsp.
multiplex
. CC6 (ALS strains) identified an
X. fastidiosa
subsp.
multiplex
-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in
X. fastidiosa
is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci
holC
,
nuoL
,
leuA
,
gltT
,
cysG
,
petC
, and
lacF
were chosen to form the basis of a public database for typing
X. fastidiosa
(
www.mlst.net
). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.
Structure and Biosynthesis of the BT Peptide Antibiotic from
Brevibacillus texasporus
Authors: Wu Xiaofeng , Ballard Johnathan , Jiang Yi Wei
Published: December 2005

We isolated a novel gram-positive bacterium,
Brevibacillus texasporus
, that produces an antibiotic, BT. BT is a group of related peptides that are produced by
B. texasporus
cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and
d
-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me
2
-4-methyl-4-[(E)-2-butenyl]-4,
N
-methyl-threonine-L-
d
O-I-V-V-
d
K-V-
d
L-K-
d
Y-L-V-CH
2
OH. In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.
Antagonistic Interactions among Marine Bacteria Impede the Proliferation of
Vibrio cholerae
Authors: Long Richard A. , Rowley David C. , Zamora Eric , Liu Jiayuan , Bartlett Douglas H. , Azam Farooq
Published: December 2005

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases.
Vibrio cholerae
is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of
V. cholerae
during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of
V. cholerae
in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of
V. cholerae
on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of
V. cholerae
inhibition than free-living bacteria.
Vibrio cholerae
was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit
V. cholerae
colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of
V. cholerae
under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of
V. cholerae
on marine particles.
Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere
Authors: DeAngelis Kristen M. , Ji Pingsheng , Firestone Mary K. , Lindow Steven E.
Published: December 2005

The nitrate-regulated promoter of
narG
in
Escherichia coli
was fused to promoterless ice nucleation (
inaZ
) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of
narG
is normally nitrate responsive only under anaerobic conditions, the L28H-
fnr
gene was provided in
trans
to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both
E. coli
DH5α and
Enterobacter cloacae
EcCT501R.
E. cloacae
and
E. coli
cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of
E. cloacae
cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in
E. cloacae
EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 μM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 μM. In the rhizosphere of wild oat (
Avena fatua
), the whole-cell bioreporter
E
.
cloacae
(pNgfp) or
E. cloacae
(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the
E. cloacae
(pNgfp) reporter in the
A. fatua
rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.
Cell Surface Display of Lipase in
Pseudomonas putida
KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications
Authors: Lee Seung Hwan , Lee Sang Yup , Park Byoung Chul
Published: December 2005

We developed a new cell surface display system in
Pseudomonas putida
KT2442 using OprF, an outer membrane protein of
Pseudomonas aeruginosa
, as an anchoring motif in a C-terminal deletion-fusion strategy. The
Pseudomonas fluorescens
SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of
P. putida
KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant
P. putida
KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant
Escherichia coli
displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (
R
)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in
P. putida
KT2442 and consequently for various biocatalytic applications.
Identification of Bacteria in Biofilm and Bulk Water Samples from a Nonchlorinated Model Drinking Water Distribution System: Detection of a Large Nitrite-Oxidizing Population Associated with
Nitrospira
spp
Authors: Martiny Adam C. , Albrechtsen Hans-Jørgen , Arvin Erik , Molin Søren
Published: December 2005

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 μg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with
Proteobacteria
and
Bacteriodetes
, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included
Acidobacteria
,
Nitrospirae
,
Planctomycetes
, and
Verrucomicrobia
, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (
P
= 0.027). Bacteria associated with
Nitrospira moscoviensis
were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with
Nitrospira
suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 × 10
5
to 2 × 10
5
nitrifying cells/ml in the bulk water and 3 × 10
5
cells/cm
2
on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.
Nickel and Cobalt Resistance Engineered inEscherichia coliby Overexpression of Serine Acetyltransferase from the Nickel Hyperaccumulator PlantThlaspi goesingense
Authors: Freeman John L. , Persans Michael W. , Nieman Ken , Salt David E.
Published: December 2005

The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plantThlaspi goesingensecauses enhanced nickel and cobalt resistance inEscherichia coli.Furthermore, overexpression ofT. goesingenseserine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction ofO-acetyl-l-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.
Outer Membranec-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction inGeobacter sulfurreducens
Authors: Mehta T. , Coppi M. V. , Childers S. E. , Lovley D. R.
Published: December 2005

The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides byGeobacter sulfurreducenswas investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells werec-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexahemec-type cytochrome. Transcripts foromcScould be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. TheomcSmRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream geneomcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6% amino acid sequence identity to OmcS. The other abundantc-type cytochrome sheared from the outer surface ofG. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetrahemec-type cytochrome. When eitheromcSoromcEwas deleted,G. sulfurreducenscould no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. ExpressingomcSoromcEintransrestored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated thatG. sulfurreducenscould not reduce Fe(III) oxide whenomcTwas expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced whenomcSwas expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides inG. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.
Effect ofl-Proline on Sake Brewing and Ethanol Stress inSaccharomyces cerevisiae
Authors: Takagi Hiroshi , Takaoka Miki , Kawaguchi Akari , Kubo Yoshito
Published: December 2005

During the fermentation of sake, cells ofSaccharomyces cerevisiaeare exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins.l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellularl-proline in cells ofS. cerevisiaegrown under ethanol stress. Anl-proline-accumulating laboratory strain carries a mutant allele ofPRO1,pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps ofl-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of thel-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation ofl-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting thePUT1gene, which is required forl-proline utilization, and replacing the wild-typePRO1allele with thepro1D154Nallele. The resultant strain accumulatedl-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulatel-proline to brew sake containing five times morel-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles.
Prophage-Like Elements in Bifidobacteria: Insights from Genomics, Transcription, Integration, Distribution, and Phylogenetic Analysis
Authors: Ventura Marco , Lee Ju-Hoon , Canchaya Carlos , Zink Ralf , Leahy S. , Moreno-Munoz J. A. , O'Connell-Motherway M. , Higgins D. , Fitzgerald Gerald F. , O'Sullivan Daniel J. , van Sinderen Douwe
Published: December 2005

So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus
Bifidobacterium
. In this report we analyzed three prophage-like elements that are present in the genomes of
Bifidobacterium breve
UCC 2003,
Bifidobacterium longum
NCC 2705, and
Bifidobacterium longum
DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA
Met
gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the
attB
sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a
B. longum
DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among
B. breve
strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.