bluuebunny/crossref_metadata_2025 huggingface dataset:
Ancient Heavy Metal Contamination in Soils as a Driver of Tolerant Anthyllis vulneraria Rhizobial Communities
Authors: Mohamad Roba , Maynaud Geraldine , Le Quéré Antoine , Vidal Céline , Klonowska Agnieszka , Yashiro Erika , Cleyet-Marel Jean-Claude , Brunel Brigitte
Published: October 2016

Anthyllis vulneraria
is a legume associated with nitrogen-fixing rhizobia that together offer an adapted biological material for mine-soil phytostabilization by limiting metal pollution. To find rhizobia associated with
Anthyllis
at a given site, we evaluated the genetic and phenotypic properties of a collection of 137 rhizobia recovered from soils presenting contrasting metal levels. Zn-Pb mine soils largely contained metal-tolerant rhizobia belonging to
Mesorhizobium metallidurans
or to another sister metal-tolerant species. All of the metal-tolerant isolates harbored the
cadA
marker gene (encoding a metal-efflux P
IB
-type ATPase transporter). In contrast, metal-sensitive strains were taxonomically distinct from metal-tolerant populations and consisted of new
Mesorhizobium
genospecies. Based on the symbiotic
nodA
marker, the populations comprise two symbiovar assemblages (potentially related to
Anthyllis
or
Lotus
host preferences) according to soil geographic locations but independently of metal content. Multivariate analysis showed that soil Pb and Cd concentrations differentially impacted the rhizobial communities and that a rhizobial community found in one geographically distant site was highly divergent from the others. In conclusion, heavy metal levels in soils drive the taxonomic composition of
Anthyllis-
associated rhizobial populations according to their metal-tolerance phenotype but not their symbiotic
nodA
diversity. In addition to heavy metals, local soil physicochemical and topoclimatic conditions also impact the rhizobial beta diversity.
Mesorhizobium
communities were locally adapted and site specific, and their use is recommended for the success of phytostabilization strategies based on
Mesorhizobium
-legume vegetation.

IMPORTANCE
Phytostabilization of toxic mine spoils limits heavy metal dispersion and environmental pollution by establishing a sustainable plant cover. This eco-friendly method is facilitated by the use of selected and adapted cover crop legumes living in symbiosis with rhizobia that can stimulate plant growth naturally through biological nitrogen fixation. We studied microsymbiont partners of a metal-tolerant legume,
Anthyllis vulneraria
, which is tolerant to very highly metal-polluted soils in mining and nonmining sites. Site-specific rhizobial communities were linked to taxonomic composition and metal tolerance capacity. The rhizobial species
Mesorhizobium metallidurans
was dominant in all Zn-Pb mines but one. It was not detected in unpolluted sites where other distinct
Mesorhizobium
species occur. Given the different soil conditions at the respective mining sites, including their heavy-metal contamination, revegetation strategies based on rhizobia adapting to local conditions are more likely to succeed over the long term compared to strategies based on introducing less-well-adapted strains.
Salmonella enterica Serovar Kentucky Flagella Are Required for Broiler Skin Adhesion and Caco-2 Cell Invasion
Authors: Salehi Sanaz , Howe Kevin , Lawrence Mark L. , Brooks John P. , Bailey R. Hartford , Karsi Attila
Published: October 2016

Nontyphoidal
Salmonella
strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently,
Salmonella enterica
serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one of the main factors that contribute to bacterial attachment to broiler skin. However, the precise role of flagella and the mechanism of attachment are unknown. There are two different flagellar subunits (
fliC
and
fljB
) expressed alternatively in
Salmonella enterica
serovars using phase variation. Here, by making deletions in genes encoding flagellar structural subunits (
flgK
,
fliC
, and
fljB
), and flagellar motor (
motA
), we were able to differentiate the role of flagella and their rotary motion in the colonization of broiler skin and cellular attachment. Utilizing a broiler skin assay, we demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables
Salmonella
motility, but this subunit is unable to mediate tight attachment. Deletion of the
flgK
gene prevents proper flagellar assembly, making
Salmonella
significantly less adherent to broiler skin than the wild type.
S
. Kentucky with deletions in all three structural genes,
fliC
,
fljB
, and
flgK
, as well as a flagellar motor mutant (
motA
), exhibited less adhesion and invasion of Caco-2 cells, while an
fljB
mutant was as adherent and invasive as the wild-type strain.

IMPORTANCE
In this work, we answered clearly the role of flagella in
S
. Kentucky attachment to the chicken skin and Caco-2 cells. We demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables
Salmonella
motility, but this subunit is unable to mediate strong attachment. Deletion of the
flgK
gene prevents proper flagellar assembly, making
Salmonella
significantly less adherent to broiler skin than the wild type.
S
. Kentucky with deletions in all three structural genes,
fliC
,
fljB
, and
flgK
, as well as a flagellar motor mutant (
motA
), exhibited less adhesion and invasion of Caco-2 cells, while an
fljB
mutant was as adherent and invasive as the wild-type strain. We expect these results will contribute to the understanding of the mechanisms of
Salmonella
attachment to food products.
Differences in Abundances of Total Vibrio spp., V. vulnificus, and V. parahaemolyticus in Clams and Oysters in North Carolina
Authors: Froelich B. A. , Phippen B. , Fowler P. , Noble R. T. , Oliver J. D.
Published: October 2016

Filter feeding shellfish can concentrate pathogenic bacteria, including
Vibrio vulnificus
and
Vibrio parahaemolyticus
, as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while
Vibrio
concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower
Vibrio
levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of
Vibrio
spp. in oysters and water were found to be quite different from those that were correlated with levels of
Vibrio
spp. in clams.

IMPORTANCE
This study shows that clams are a potential source of infection in North Carolina, especially for
V. parahaemolyticus
. These findings also highlight the need for clam-specific environmental research to develop accurate
Vibrio
abundance models and to broaden the ecological understanding of clam-
Vibrio
interactions. This is especially relevant as foodborne
Vibrio
infections from clams are being reported.
Overexpression of
OLE1
Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae
Authors: Fang Zhijia , Chen Zhongxiang , Wang Song , Shi Ping , Shen Yuhu , Zhang Youshang , Xiao Junhua , Huang Zhiwei
Published: October 2016

The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans.
Saccharomyces cerevisiae
is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene,
OLE1
, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of
OLE1
was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for
OLE1
). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of
OLE1
, leading to cadmium resistance. Such regulation of
OLE1
in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium.

IMPORTANCE
A
S. cerevisiae
gene encoding a delta-9 desaturase,
OLE1
, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of
OLE1
in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of
OLE1
involvement in membrane stability may indicate a novel defense strategy against cadmium stress.
Pseudo-Second-Order Calcium-Mediated Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms
Authors: Luo Xia , Jedlicka Sabrina , Jellison Kristen
Published: October 2016

Cryptosporidium parvum
oocysts are able to infect a wide range of mammals, including humans, via fecal-oral transmission. The remobilization of biofilm-associated
C. parvum
oocysts back into the water column by biofilm sloughing or bulk erosion poses a threat to public health and may be responsible for waterborne outbreaks; thus, the investigation of
C. parvum
attachment mechanisms to biofilms, particularly the physical and chemical factors controlling oocyst attachment to biofilms, is essential to predict the behavior of oocysts in the environment. In our study, biofilms were grown in rotating annular bioreactors using prefiltered stream water (0.2-μm retention) and rock biofilms (6-μm retention) until the mean biofilm thickness reached steady state. Oocyst deposition followed a calcium-mediated pseudo-second-order kinetic model. Kinetic parameters (i.e., initial oocyst deposition rate constant and total number of oocysts adhered to biofilms at equilibrium) from the model were then used to evaluate the impact of water conductivity on the attachment of oocysts to biofilms. Oocyst deposition was independent of solution ionic strength; instead, the presence of calcium enhanced oocyst attachment, as demonstrated by deposition tests. Calcium was identified as the predominant factor that bridges the carboxylic functional groups on biofilm and oocyst surfaces to cause attachment. The pseudo-second-order kinetic profile fit all experimental conditions, regardless of water chemistry and/or lighting conditions.

IMPORTANCE
The cation bridging model in our study provides new insights into the impact of calcium on the attachment of
C. parvum
oocysts to environmental biofilms. The kinetic parameters derived from the model could be further analyzed to elucidate the behavior of oocysts in commonly encountered complex aquatic systems, which will enable future innovations in parasite detection and treatment technologies to protect public health.
Exploring the Impacts of Postharvest Processing on the Microbiota and Metabolite Profiles during Green Coffee Bean Production
Authors: De Bruyn Florac , Zhang Sophia Jiyuan , Pothakos Vasileios , Torres Julio , Lambot Charles , Moroni Alice V. , Callanan Michael , Sybesma Wilbert , Weckx Stefan , De Vuyst Luc
Published: October 2016

The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e.,
Leuconostoc
,
Lactococcus
, and
Lactobacillus
) was the most commonly identified microbial group, along with enterobacteria and yeasts (
Pichia
and
Starmerella
). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e.,
Acetobacter
and
Gluconobacter
) were most abundant, along with
Pichia
and non-
Pichia
(
Candida
,
Starmerella
, and
Saccharomycopsis
) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process.

IMPORTANCE
Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.
Bacteria as Emerging Indicators of Soil Condition
Authors: Hermans Syrie M. , Buckley Hannah L. , Case Bradley S. , Curran-Cournane Fiona , Taylor Matthew , Lear Gavin
Published: October 2016

Bacterial communities are important for the health and productivity of soil ecosystems and have great potential as novel indicators of environmental perturbations. To assess how they are affected by anthropogenic activity and to determine their ability to provide alternative metrics of environmental health, we sought to define which soil variables bacteria respond to across multiple soil types and land uses. We determined, through 16S rRNA gene amplicon sequencing, the composition of bacterial communities in soil samples from 110 natural or human-impacted sites, located up to 300 km apart. Overall, soil bacterial communities varied more in response to changing soil environments than in response to changes in climate or increasing geographic distance. We identified strong correlations between the relative abundances of members of
Pirellulaceae
and soil pH, members of
Gaiellaceae
and carbon-to-nitrogen ratios, members of
Bradyrhizobium
and the levels of Olsen P (a measure of plant available phosphorus), and members of
Chitinophagaceae
and aluminum concentrations. These relationships between specific soil attributes and individual soil taxa not only highlight ecological characteristics of these organisms but also demonstrate the ability of key bacterial taxonomic groups to reflect the impact of specific anthropogenic activities, even in comparisons of samples across large geographic areas and diverse soil types. Overall, we provide strong evidence that there is scope to use relative taxon abundances as biological indicators of soil condition.

IMPORTANCE
The impact of land use change and management on soil microbial community composition remains poorly understood. Therefore, we explored the relationship between a wide range of soil factors and soil bacterial community composition. We included variables related to anthropogenic activity and collected samples across a large spatial scale to interrogate the complex relationships between various bacterial community attributes and soil condition. We provide evidence of strong relationships between individual taxa and specific soil attributes even across large spatial scales and soil and land use types. Collectively, we were able to demonstrate the largely untapped potential of microorganisms to indicate the condition of soil and thereby influence the way that we monitor the effects of anthropogenic activity on soil ecosystems into the future.
Genome Sequence of Paraburkholderia nodosa Strain CNPSo 1341, a N
2
-Fixing Symbiont of the Promiscuous Legume Phaseolus vulgaris
Authors: Dall'Agnol Rebeca Fuzinatto , Costa Maira Rejane , Ribeiro Renan Augusto , Delamuta Jakeline Renata Marçon , Chueire Ligia Maria Oliveira , Hungria Mariangela
Published: November 2016

Paraburkholderia nodosa
CNPSo 1341 is a N
2
-fixing symbiont of
Phaseolus vulgaris
isolated from an undisturbed soil of the Brazilian Cerrado. Its draft genome contains 8,614,032 bp and 8,068 coding sequences (CDSs). Nodulation and N
2
-fixation genes were clustered in the genome that also contains several genes of secretion systems and quorum sensing.
Draft Genome Sequence of a Hospital-Associated Clone of Klebsiella pneumoniae ST340/CC258 Coproducing RmtG and KPC-2 Isolated from a Pediatric Patient
Authors: Cerdeira Louise , Fernandes Miriam R. , Francisco Gabriela R. , Bueno Maria Fernanda C. , Ienne Susan , Souza Tiago A. , de Oliveira Garcia Doroti , Lincopan Nilton
Published: November 2016

We report here the draft genome sequence of a
Klebsiella pneumoniae
strain 1194/11, belonging to the hospital-associated sequence type 340 (ST340; clonal complex CC258), isolated from a catheter tip culture from a pediatric patient. The multidrug-resistant strain coproduced the 16S rRNA methyltransferase rRNA RmtG and β-lactamases KPC-2 and CTX-M-15.
Draft Genome Sequence of the Bacterium Comamonas aquatica CJG
Authors: Dai Wenkui , Zhu Yutong , Wang Xiqian , Sakenova Nazgul , Yang Zhenyu , Wang Hongjuan , Li Guoqiao , He Jiajing , Huang Deyu , Cai Yongfei , Guo Weizheng , Wang Qian , Feng Tingting , Fan Quanshui , Zheng Tianling , Han Aidong
Published: November 2016

A Gram-negative bacterial strain,
Comamonas aquatica
CJG, absorbs low-density lipoprotein but not high-density lipoprotein in serum. Here, we report its draft genomic sequence of 3,764,434 bp, containing total 3,425 genes, 27% of which encode proteins for metabolism and energy conversion, and it is 30% identical to the genome of
Comamonas testosteroni
.
Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom
Authors: Zeng Zhiwei , Zhang Jing , Jing Shuping , Cheng Zetao , Bofill-Mas Silvia , Maluquer de Motes Carlos , Hundesa Ayalkibet , Girones Rosina , Seto Donald , Zhang Qiwei
Published: November 2016

The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.
Genome Sequence of
Prosthecochloris
sp. Strain CIB 2401 of the Phylum
Chlorobi
Authors: Nabhan Shaza , Bunk Boyke , Spröer Cathrin , Liu Zhenfeng , Bryant Donald A. , Overmann Jörg
Published: November 2016

To date, only 13 genomes of green sulfur bacteria (family
Chlorobiaceae
) have been sequenced. The sequenced strains do not cover the full phylogenetic diversity of the family. We determined the complete genome sequence of
Prosthecochloris
sp. strain CIB 2401, thereby increasing the genome information for the poorly represented marine
Chlorobiaceae
.
marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli
Authors: Cohen S P , McMurry L M , Levy S B
Published: November 2016

Mar (multiple antibiotic resistant) mutants of Escherichia coli express chromosomally mediated resistance to a variety of structurally unrelated hydrophilic and hydrophobic antibiotics. Insertion of transposon Tn5 into the marA locus at min 34.05 on the chromosome completely reverses the Mar phenotype (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983). We found that among changes in the outer membrane of Mar mutants, porin OmpF was greatly reduced, although Mar mutants were more resistant than cells lacking only OmpF. Transduction of the marA region from a Mar strain, but not a wild-type strain, led to loss of OmpF. P1 transduction of marA::Tn5 into a Mar mutant partially restored OmpF levels. Therefore, OmpF reduction required a mutation in the marA region. Mar mutants of an ompF-lacZ operon fusion strain expressed 50 to 75% of the beta-galactosidase activity of the isogenic non-Mar parental strain, while Mar mutants of a protein fusion strain expressed less than 10% of the enzyme activity in the non-Mar strain. These changes were completely reversed by insertion of marA::Tn5. The responsiveness of OmpF-LacZ to osmolarity and temperature changes was similar in Mar and wild-type strains. Although some transcriptional control may have been present, OmpF reduction appeared to occur primarily by a posttranscriptional mechanism. The steady-state levels of ompF mRNA were twofold lower and the mRNA was five times less stable in the Mar mutant than in the wild-type strain. Expression of micF, which lowers ompF mRNA levels, was elevated in Mar strains, as revealed by a micF-lacZ fusion. Studies with strains deleted for the micF locus showed that the marA-dependent reduction of OmpF required an intact micF locus. Our findings suggest that the marA locus directly or indirectly increases micF expression, causing a posttranscriptional decrease in ompF mRNA and reduced amounts of OmpF.
Regulation of gene expression in plasmid ColE1: delayed expression of the kil gene
Authors: Zhang S P , Yan L F , Zubay G
Published: November 2016

cea, imm, and kil are a cluster of three functionally related genes of the plasmid ColE1. The cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene. The imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction. Complementary interaction between the imm mRNA and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene when the cea-kil operon is induced. A segment in the overlapping region between the cea and imm genes causes delayed expression of the kil gene in the absence of imm gene transcription. This delay effect increases the yields of colicin synthesized in induced cells.
Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii
Authors: Kirtland G M , Morris T D , Moore P H , O'Brian J J , Edmonds C G , McCloskey J A , Katze J R
Published: November 2016

Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.
Role of the 5' upstream sequence and tandem promoters in regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon
Authors: Nesin M , Lupski J R , Godson G N
Published: November 2016

Bal31 exonuclease deletion analysis and transposon Tn5 mutagenesis of the 5' regulatory region of the rpsU-dnaG-rpoD macromolecular synthesis operon fused to the chloramphenicol acetyltransferase gene (pGLR301) demonstrated that sequences 5' to the operon promoters were not involved in operon transcriptional regulation and that the three tandem promoters P1, P2, and P3 were functionally independent. P2 was the strongest promoter, and P3 was the weakest. P1, P2, and P3 acting in combination appeared to be stronger than the individual promoters.
Analysis of recombination occurring at SLP1 att sites
Authors: Lee S C , Omer C A , Brasch M A , Cohen S N
Published: November 2016

SLP1int is a conjugative Streptomyces coelicolor genetic element that can transfer to Streptomyces lividans and integrate site specifically into the genome of the new bacterial host. Recombination of SLP1 previously has been shown to occur within nearly identical 112-base-pair att sequences on the plasmid and host chromosome. We report here that both integrative recombination and intermolecular transfer of SLP1int require no more than a 48-base-pair segment of the att sequence and that SLP1 transfer occurs by a conservative rather than a replicative mechanism. The functions responsible for the excision of the element as a discrete DNA segment are induced during the conjugal transfer of SLP1.
Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis
Authors: Francesconi S C , MacAlister T J , Setlow B , Setlow P
Published: November 2016

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.
Lysine biosynthesis pathway and biochemical blocks of lysine auxotrophs of Schizosaccharomyces pombe
Authors: Ye Z H , Bhattacharjee J K
Published: November 2016

The alpha-aminoadipate (AA) pathway for the biosynthesis of lysine was investigated in the wild type and in lysine auxotrophs of the fission yeast Schizosaccharomyces pombe. Of the eight enzyme activities of the AA pathway that have been examined so far, six were present in the extract of wild-type S. pombe cells. Growth response to AA and accumulation studies indicated that three lysine auxotrophs, the lys2-97, lys4-95, and lys8-1 strains, were blocked before the AA step and that four lysine auxotrophs, the lys1-131, lys3-37, lys6-3, and lys7-2 strains, were blocked after the AA step. Among the mutants investigated, the lys2-97 mutant exhibited an enzyme lesion at the cis-homoaconitate hydratase step, the lys1-131 and lys7-2 mutants exhibited lesions at the AA reductase step, and lys3-37 exhibited a lesion at the saccharopine dehydrogenase step. These results demonstrated the basic similarity of the AA pathway in S. pombe and Saccharomyces cerevisiae.
Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement
Authors: Collins C M , Falkow S
Published: November 2016

Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at least 3.2 kilobases of this fragment were necessary for production of urease. The urease recombinant plasmid pURE coded for at least four insert-specific polypeptides as determined by maxicell analysis. Disruption of the region encoding two of these polypeptides (67 and 27 kilodaltons) abolished urease activity. Analysis by Southern hybridization of urease-positive E. coli 1021 and seven independently isolated urease-negative segregants showed that a DNA rearrangement was associated with the urease-negative phenotype.
Turgor pressure responses of a gram-negative bacterium to antibiotic treatment, measured by collapse of gas vesicles
Authors: Pinette M F , Koch A L
Published: November 2016

The internal hydrostatic pressure of Ancylobacter aquaticus was measured by collapsing the gas vesicles with an externally applied pressure. Turgor pressure was measured in conjunction with various antibiotic treatments to elucidate some aspects of the biophysics of gram-negative cell wall function. Differences in the effects of these drugs either alone or in combination with other treatments were related to known biochemical activities of these drugs. Our previous work, demonstrating a heterogeneous cellular response to beta-lactam antibodies, was confirmed and extended. Most of the cell wall growth-inhibiting antibiotics resulted in some cells (those in component I) developing a higher pressure, while the remainder (those in component II) lost turgor. Although the fraction of the cells in each component varied a little from subculture to subculture, it did not vary with time or choice of antibiotic treatment. Mecillinam gave a nearly monophasic response. All antibiotics blocking macromolecular synthesis gave monophasic curves. The 50% collapse pressure in some cases, however, was lower higher, or the same as the control.
Genomic instability in Rhizobium phaseoli
Authors: Flores M , González V , Pardo M A , Leija A , Martínez E , Romero D , Piñero D , Dávila G , Palacios R
Published: November 2016

Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties. On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements. In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations. DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements. The implications and possible biological significance of these observations are discussed.
A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli
Authors: Vodkin M H , Williams J C
Published: November 2016

A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen.
Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula
Authors: Ojha M , Wallace C J
Published: November 2016

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.
Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila
Authors: Gobius K S , Pemberton J M
Published: November 2016

The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.
Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations
Authors: Garges S , Adhya S
Published: November 2016

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.
Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6
Authors: McBride K E , Knauf V C
Published: November 2016

The virE operon of the Agrobacterium tumefaciens Ti plasmid pTiA6 encodes at least one trans-acting protein involved in the expression of virulence. Two open reading frames designated virE1 and virE2 code for polypeptides of 7 and 60 kilodaltons (kDa), respectively, that can be visualized after expression in Escherichia coli minicells. To determine which virE sequences are required for virulence, a strain deleted for the entire locus [strain KE1(pTiA6 delta E)] was constructed and tested for the ability to be complemented by subclones with and without site-directed mutations in the virE operon. One subclone containing only virE1 and virE2 as well as upstream promoter sequences was sufficient to restore full virulence on the host plant Kalanchoe daigremontiana. However, some other virulence locus representing a host range determinant appeared to be deleted from strain KE1(pTiA6 delta E), since virE1 and virE2 were not sufficient to fully restore virulence on wounded tomato plants. virE operon constructs with specific lesions in either virE1 or virE2 were impaired for complementation of pTiA6 delta E. Several mutations specific for the promoter-proximal virE1 locus appeared to have a polar effect on expression of the virE2-encoded 60-kDa protein. However, virE2::lacZ fusion constructs suggest that this effect is not at the level of transcription or translation. Collectively, these data indicate that both the 7- and the 60-kDa polypeptides are virulence determinants for the Ti plasmid pTiA6 and suggest that the 60-kDa protein may be less stable in the absence of the 7-kDa protein.
Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis
Authors: Fosnaugh K , Greenberg E P
Published: November 2016

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.
Absence of autolytic activity (peptidoglycan nicking) in penicillin-induced nonlytic death in a group A streptococcus
Authors: McDowell T D , Lemanski C L
Published: November 2016

The extent of sublytic autolysin activity (peptidoglycan [PG] nicking) after exposure of exponentially growing cultures of a group A streptococcus (GAS) to benzylpenicillin (PenG) was studied by determining changes in the glycan chain length of PG polymers. The average PG chain length in isolated cell walls was estimated by calculating the ratio of the total hexosamine content (Morgan-Elson-reactive material) to reducing-end group content established via quantitation of [3H]borohydride reduction products. Comparison of the average PG chain length obtained from untreated control cultures of GAS with those obtained after exposure to a saturating dose of PenG revealed no decrease over a time interval equivalent to four mass doublings of the control cultures. Exposure to this concentration of PenG for a time equivalent to only two mass doublings resulted in approximately 90% loss of viability. In contrast, exposure of the lytic bacterium, Streptococcus faecium ATCC 9790, to a 50% growth inhibitory dose of PenG produced a 20% reduction in the average PG chain length concomitant with only a 65% loss of viability. Preliminary characterization of the autolytic system of GAS indicated that this streptococcus has a hexosaminidase-type autolysin. The results presented indicate the lack of autolytic activity in PenG-induced nonlytic death.
Restriction analysis and quantitative estimation of methylated bases of filamentous and unicellular cyanobacterial DNAs
Authors: Padhy R N , Hottat F G , Coene M M , Hoet P P
Published: November 2016

The DNAs of strains of three cyanobacterial genera (Anabaena, Plectonema, and Synechococcus) were found to be partially or fully resistant to many restriction endonucleases. This could be due to the absence of specific sequences or to modifications, rendering given sequences resistant to cleavage. The latter explanation is substantiated by the content of N6-methyladenine and 5-methylcytosine in these genomes, which is high in comparison with that in other bacterial genomes. dcm- and dam-like methylases are present in the three strains (based on the restriction patterns obtained with the appropriate isoschizomeric enzymes). Their contribution to the overall content of methyladenine and methylcytosine in the genomes was calculated. Partial methylation of GATC sequences was observed in Anabaena DNA. In addition, the GATC methylation patterns might not have been random in the three cyanobacterial DNA preparations, as revealed by the appearance of discrete fragments (possibly of plasmid origin) withstanding cleavage by DpnI (which requires the presence of methyladenine in the GATC sequence).
Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein
Authors: Hanson M S , Hempel J , Brinton C C
Published: November 2016

Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1.
Structure and organization of the pel genes from Erwinia chrysanthemi EC16
Authors: Tamaki S J , Gold S , Robeson M , Manulis S , Keen N T
Published: November 2016

The pelA and pelC genes from Erwinia chrysanthemi EC16 were sequenced and overexpressed in Escherichia coli cells. These genes and two others from the same strain that were characterized previously encode catalytically related pectate lyase proteins that are involved with the maceration and soft-rotting of plant tissue. The pel genes of strain EC16 were organized as two loosely linked clusters, with two structurally homologous genes in each. The pelA/E cluster also contained the remains of an additional pel gene, the 5' portion of which had been removed by a prior deletion event. Each of the four functional pel genes but not the deleted one contained an efficient rho-independent transcriptional terminator after the translational stop. These and other data indicate that the pel genes are all independently regulated despite their structural homology and tandem clustered organization. Two of the genes, pelA and pelE, encoded proteins that differed greatly in their isoelectric points and ability to macerate plant tissue. A recombinant gene constructed with the 5' portion of pelE and the 3' portion of pelA yielded a chimeric protein with high pectate lyase activity but relatively low maceration activity. This result raised the possibility that the poor maceration ability of the pelA gene product may involve other properties in addition to its low isoelectric point.
Molecular characterization of the type 3 (MR/K) fimbriae of Klebsiella pneumoniae
Authors: Gerlach G F , Allen B L , Clegg S
Published: November 2016

The expression of type 3 (MR/K) fimbriae by Klebsiella pneumoniae requires the production of at least four polypeptides with molecular masses of 20.5, 25, 34, and 78 kilodaltons. The genes encoding these polypeptides are located on a gene cluster 5,500 base pairs in length. The nucleotide sequence of the major fimbrial structural gene was determined, and its transcription was shown to initiate in vitro 157 base pairs upstream of the translational start site. The predicted amino acid sequence for the fimbrial subunit comprised 202 residues, including a signal peptide of 22 amino acids. Despite similarities in the organization of the gene cluster, the nucleotide and the amino acid sequence of the major fimbrial subunit revealed little agreement with other known fimbrial subunit sequences.
Purification and characterization of salmolysin, an extracellular hemolytic toxin from Aeromonas salmonicida
Authors: Nomura S , Fujino M , Yamakawa M , Kawahara E
Published: November 2016

An extracellular hemolytic toxin of Aeromonas salmonicida, termed salmolysin, was purified 945-fold by ammonium sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and gel filtration chromatography on Sephadex G-100 and Sepharose 2B. Salmolysin appeared homogeneous upon cellulose acetate membrane electrophoresis and immunodiffusion analysis. The molecular weight of the toxin was estimated to be approximately 200,000 by the sedimentation equilibrium method. The UV absorption spectrum showed a maximum at 275 nm and a minimum at 262 nm. The isoelectric point was found to be at pI 5.4. Carbohydrate and protein analyses and other biochemical data indicated that salmolysin is a glycoprotein, containing approximately 62% carbohydrates. The toxin is a heat-labile substance and is stable at a neutral pH value. Ferrous ion inhibited the activity, whereas metal-chelating agents did not affect the activity. Sulfhydryl reagents did not inhibit the toxin, whereas reducing agents, such as L-cysteine and reduced glutathione, inhibited the toxin to a certain extent. Salmolysin was inactivated by a nonionic detergent but was stimulated by an anionic detergent, sodium deoxycholate, at a low concentration. The toxin was also inactivated by subtilisin and trypsin but was not inhibited by papain and pepsin. Salmolysin, with a remarkable hemolytic activity against salmonid erythrocytes, was lethal to rainbow trout when it was injected intramuscularly.