bluuebunny/crossref_metadata_2025 huggingface dataset:
High Abundances of Aerobic Anoxygenic Photosynthetic Bacteria in the South Pacific Ocean
Authors: Lami Raphaël , Cottrell Matthew T. , Ras Joséphine , Ulloa Osvaldo , Obernosterer Ingrid , Claustre Hervé , Kirchman David L. , Lebaron Philippe
Published: May 2007

Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria,
Prochlorococcus
, and total prokaryotic abundances, as well as bacteriochlorophyll
a
(BChl
a
) and divinyl-chlorophyll
a
concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and
Prochlorococcus
were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 × 10
5
cells ml
−1
and as high as 24% of the overall community. These measurements were consistent with the high BChl
a
concentrations (up to 3.32 × 10
−3
μg liter
−1
) found at all stations. However, the BChl
a
content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock.
Development and Application of Real-Time PCR Assays for Quantification of
erm
Genes Conferring Resistance to Macrolides-Lincosamides-Streptogramin B in Livestock Manure and Manure Management Systems
Authors: Chen Jing , Yu Zhongtang , Michel Frederick C. , Wittum Thomas , Morrison Mark
Published: May 2007

Erythromycin and tylosin are commonly used in animal production, and such use is perceived to contribute to the overall antimicrobial resistance (AR) reservoirs. Quantitative measurements of this type of AR reservoir in microbial communities are required to understand AR ecology (e.g., emergence, persistence, and dissemination). We report here the development, validation, and use of six real-time PCR assays for quantifying six classes of
erm
genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamides-streptogramin B (MLS
B
). These real-time PCR assays were validated and used in quantifying the six
erm
classes in five types of samples, including those from bovine manure, swine manure, compost of swine manure, swine waste lagoons, and an Ekokan upflow biofilter system treating hog house effluents. The bovine manure samples were found to contain much smaller reservoirs of each of the six
erm
classes than the swine manure samples. Compared to the swine manure samples, the composted swine manure samples had substantially reduced
erm
gene abundances (by up to 7.3 logs), whereas the lagoon or the biofilter samples had similar
erm
gene abundances. These preliminary results suggest that the methods of manure storage and treatment probably have a substantial impact on the persistence and decline of MLS
B
resistance originating from food animals, thus likely affecting the dissemination of such resistance genes into the environment. The abundances of these
erm
genes appeared to be positively correlated with those of the
tet
genes determined previously among these samples. These real-time PCR assays provide a rapid, quantitative, and cultivation-independent measurement of six major classes of
erm
genes, which should be useful for ecological studies of AR.
The
Schizosaccharomyces pombe
Cdc7 Protein Kinase Required for Septum Formation Is a Client Protein of Cdc37
Authors: Liang Jun , Fantes Peter
Published: May 2007

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive
cdc37
alleles in
S. pombe
and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a
cdc37ts
mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated
cdc7
gene was identified. Further experiments showed that the mutation in this strain was indeed in
cdc7
. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the
cdc37ts
mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.
Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored α-Glucanotransferase Enzymes ofAspergillus niger
Authors: van der Kaaij R. M. , Yuan X.-L. , Franken A. , Ram A. F. J. , Punt P. J. , van der Maarel M. J. E. C. , Dijkhuizen L.
Published: May 2007

In the genome sequence ofAspergillus nigerCBS 513.88, three genes were identified with high similarity to fungal α-amylases. The protein sequences derived from these genes were different in two ways from all described fungal α-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the α-amylase family were absent. We expressed two of these enzymes in a suitableA. nigerstrain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with α-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new α-(1,4)-glycosidic bonds and therefore belong to the group of the 4-α-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing α-(1,3)-glycosidic bonds as acceptor substrate. AnagtAknockout ofA. nigershowed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with α-glucans in their cell walls, but not in yeast species lacking cell wall α-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.
Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in
Blastomyces dermatitidis
Illuminates the Role of Septin in Morphogenesis and Sporulation
Authors: Krajaejun T. , Gauthier G. M. , Rappleye C. A. , Sullivan T. D. , Klein B. S.
Published: May 2007

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus
Blastomyces dermatitidis
, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to
GFP
to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in
B. dermatitidis
, we first used
LACZ
and the virulence gene
BAD1
as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A
CDC11
septin homolog in
B. dermatitidis
localized to the neck of budding yeast cells.
CDC11
-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of
B. dermatitidis
.
Temperature-Hypersensitive Sites of the Flagellar Switch Component FliG in
Salmonella enterica
Serovar Typhimurium
Authors: Mashimo Takuji , Hashimoto Manami , Yamaguchi Shigeru , Aizawa Shin-Ichi
Published: May 2007

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla
−
, Mot
−
, and Che
−
), depending on the sites and types of mutations. Some of the Mot
−
mutants previously characterized are found to be motile. Reexamination of all Mot
−
mutants in
fliGMN
genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20°C but nonmotile at 37°C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of
fliM
and
fliN
and most of the
fliG
TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the
fliG
mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.
DNA Polymerases BI and D from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs
Authors: Tori Kazuo , Kimizu Megumi , Ishino Sonoko , Ishino Yoshizumi
Published: May 2007

Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication.
Pyrococcus furiosus
, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal
P. furiosus
PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.
Horizontal Gene Transfer and Homologous Recombination Drive the Evolution of the Nitrogen-Fixing Symbionts ofMedicagoSpecies
Authors: Bailly Xavier , Olivieri Isabelle , Brunel Brigitte , Cleyet-Marel Jean-Claude , Béna Gilles
Published: May 2007

Using nitrogen-fixingSinorhizobiumspecies that interact withMedicagoplants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genusMedicagoon the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact withMedicagospecies, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, usingMedicago truncatulaandMedicago laciniataas host plants, to study the differentiation between populations ofSinorhizobium medicae,Sinorhizobium melilotibv. meliloti, andS. melilotibv. medicaginis. The former two can be associated withM. truncatula(among other species ofMedicago), whereas the last organism is the specific symbiont ofM. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids ofS. meliloti.The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification ofMedicagosymbionts. WithinS. meliloti, the analyses show thatnodgenes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars ofS. meliloti, whereas reproductive isolation betweenS. melilotibv. meliloti andS. medicaeis maintained even though these bacteria can cooccur in sympatry on the same individual host plants.
Target Genes and DNA-Binding Sites of the Response Regulator PhoR fromCorynebacterium glutamicum
Authors: Schaaf Sarah , Bott Michael
Published: May 2007

The two-component signal transduction system PhoRS ofCorynebacterium glutamicumis involved in the phosphate (Pi) starvation response. To analyze the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible (psi) genes, this response regulator and the kinase domain of its cognate sensor, PhoS (MBP-PhoSΔ1-246), were overproduced and purified. MBP-PhoSΔ1-246 showed constitutive autophosphorylation activity, and a rapid phosphoryl group transfer from phosphorylated MBP-PhoSΔ1-246 to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR. The affinity of PhoR∼P to different promoters varied and decreased in the orderpstSCAB>phoRS>phoC>ushA>porB>ugpA>pitA>nucHandphoH1>glpQ1. The binding sites in front ofpstSCABandphoRSwere localized at positions −194 to −176 and −61 to −43 upstream of the transcriptional start sites, respectively. Alignment of these two 19-bp binding sites revealed a high identity in the 5′-terminal part, but not in the 3′-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by 3 bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which might represent PhoR binding sites. The in vivo relevance of the PhoR-binding site within thephoRSpromoter was supported by reporter gene studies.
CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from EnterotoxigenicEscherichia coli
Authors: Pilonieta M. Carolina , Bodero Maria D. , Munson George P.
Published: May 2007

H10407 is a strain of enterotoxigenicEscherichia coli(ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promotercfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site ofcfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene,cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, andcexEp::lacZreporters demonstrated that CfaD is required for the expression ofcexEin vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.
Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) ofPseudomonas aeruginosa
Authors: Sakuragi Yumiko , Kolter Roberto
Published: May 2007

Quorum sensing (QS) has been previously shown to play an important role in the development ofPseudomonas aeruginosabiofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of thepeloperon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced inlasIandrhlImutants. Chemical complementation of thelasImutant with 3-oxo-dodecanoyl homoserine lactone restorespeltranscription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.
The
Haemophilus influenzae
hFbpABC Fe
3+
Transporter: Analysis of the Membrane Permease and Development of a Gallium-Based Screen for Mutants
Authors: Anderson Damon S. , Adhikari Pratima , Weaver Katherine D. , Crumbliss Alvin L. , Mietzner Timothy A.
Published: May 2007

The obligate human pathogen
Haemophilus influenzae
utilizes a siderophore-independent (free) Fe
3+
transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe
3+
through the periplasm and across the inner membrane of
H. influenzae
. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient
Escherichia coli
background, and effects of mutations were analyzed using growth rescue and radiolabeled
55
Fe
3+
transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.
Identification of Two
Mycobacterium smegmatis
Lipoproteins Exported by a SecA2-Dependent Pathway
Authors: Gibbons Henry S. , Wolschendorf Frank , Abshire Michelle , Niederweis Michael , Braunstein Miriam
Published: May 2007

The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and Δ
secA2
mutant
Mycobacterium smegmatis
, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous
M. smegmatis
proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of
Mycobacterium tuberculosis
, as indicated by the appearance of an export intermediate when the protein was expressed in a Δ
secA2
mutant of
M. tuberculosis
. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between
M. smegmatis
and
M. tuberculosis.
Evidence for Modified Mechanisms of Chloroethene Oxidation in
Pseudomonas butanovora
Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase
Authors: Halsey Kimberly H. , Doughty David M. , Sayavedra-Soto Luis A. , Bottomley Peter J. , Arp Daniel J.
Published: May 2007

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of
Pseudomonas butanovora
containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-
cis
-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-
cis
-DCE epoxide during oxidation of 1,2-
cis
-DCE, only about 50% of the 1,2-
cis
-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-
cis
-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-
cis
-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O
2
uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with
P. butanovora
BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.
A Functional Homing Endonuclease in the
Bacillus anthracis nrdE
Group I Intron
Authors: Nord David , Torrents Eduard , Sjöberg Britt-Marie
Published: May 2007

The essential
Bacillus anthracis nrdE
gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the
nrdE
pre-mRNA is spliced and that the homing endonuclease cleaves an intronless
nrdE
gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3′ extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly,
nrdE
genes from several other
Bacillaceae
were also susceptible to cleavage, with those of
Bacillus cereus
,
Staphylococcus epidermidis
(
nrdE1
),
B. anthracis
, and
Bacillus thuringiensis
serovar konkukian being better substrates than those of
Bacillus subtilis
,
Bacillus lichenformis
, and
S. epidermidis
(
nrdE2
). On the other hand,
nrdE
genes from
Lactococcus lactis
,
Escherichia coli
,
Salmonella enterica
serovar Typhimurium, and
Corynebacterium ammoniagenes
were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase
nrdE
gene is a frequent target for self-splicing IVSs. A comparison of
nrdE
genes from seven gram-positive low-G+C bacteria, two bacteriophages, and
Nocardia farcinica
showed five different insertion sites for self-splicing IVSs within the coding region of the
nrdE
gene.
Only One of Four Oligopeptide Transport Systems Mediates Nitrogen Nutrition inStaphylococcus aureus
Authors: Hiron Aurelia , Borezée-Durant Elise , Piard Jean-Christophe , Juillard Vincent
Published: May 2007

Oligopeptides internalized by oligopeptide permease (Opp) transporters play key roles in bacterial nutrition, signaling, and virulence. To date, twooppoperons,opp-1andopp-2, have been identified inStaphylococcus aureus. Systematic in silico analysis of 11 differentS. aureusgenomes revealed the existence of two newoppoperons,opp-3andopp-4, plus anopp-5Agene encoding a putative peptide-binding protein. With the exception ofopp-4, theoppoperons were present in allS. aureusstrains. Within a single strain, the differentoppoperons displayed little sequence similarity and distinct genetic organization. Transcriptional studies showed thatopp-1,opp-2,opp-3, andopp-4operons were polycistronic and thatopp-5Ais monocistronic. We designed a minimal chemically defined medium forS. aureusRN6390 and showed that alloppgenes were expressed but at different levels. Where tested, OppA protein production paralleled transcriptional profiles.opp-3, which encodes proteins most similar to known peptide transport proteins, displayed the highest expression level and was the only transporter to be regulated by specific amino acids, tyrosine and phenylalanine. Defined deletion mutants in one or several peptide permeases were constructed and tested for their capacity to grow in peptide-containing medium. Among the four putative Opp systems, Opp-3 was the only system able to provide oligopeptides for growth, ranging in length from 3 to 8 amino acids. Dipeptides were imported exclusively by DtpT, a proton-driven di- and tripeptide permease. These data provide a first complete inventory of the peptide transport systemsoppanddtpTofS. aureus. Among them, the newly identified Opp-3 appears to be the main Opp system supplying the cell with peptides as nutritional sources.
VapC-1 of Nontypeable
Haemophilus influenzae
Is a Ribonuclease
Authors: Daines Dayle A. , Wu Mack H. , Yuan Sarah Y.
Published: May 2007

Nontypeable
Haemophilus influenzae
(NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the
vapBC
family. Here, we have characterized the function of one of the NTHi alleles,
vapBC-1
. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an
Escherichia coli
expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both
E. coli
and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi
vapBC-1
gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.
Octanoyl-Homoserine Lactone Is the Cognate Signal for
Burkholderia mallei
BmaR1-BmaI1 Quorum Sensing
Authors: Duerkop Breck A. , Ulrich Ricky L. , Greenberg E. Peter
Published: May 2007

Acyl-homoserine lactones (HSLs) serve as quorum-sensing signals for many
Proteobacteria
. Members of the LuxI family of signal generators catalyze the production of acyl-HSLs, which bind to a cognate receptor in the LuxR family of transcription factors. The obligate animal pathogen
Burkholderia mallei
produces several acyl-HSLs, and the
B. mallei
genome has four
luxR
and two
luxI
homologs, each of which has been established as a virulence factor. To begin to delineate the relevant acyl-HSL signals for
B. mallei
LuxR homologs, we analyzed the BmaR1-BmaI1 system. A comparison of acyl-HSL profiles from
B. mallei
ATCC 23344 and a
B. mallei bmaI1
mutant indicates that octanoyl-HSL synthesis is BmaI1 dependent. Furthermore, octanoyl-HSL is the predominant acyl-HSL produced by BmaI1 in recombinant
Escherichia coli
. The synthesis of soluble BmaR1 in recombinant
E. coli
requires octanoyl-HSL or decanoyl-HSL. Insoluble aggregates of BmaR1 are produced in the presence of other acyl-HSLs and in the absence of acyl-HSLs. The
bmaI1
promoter is activated by BmaR1 and octanoyl-HSL, and a 20-bp inverted repeat in the
bmaI1
promoter is required for
bmaI1
activation. Purified BmaR1 binds to this promoter region. These findings implicate octanoyl-HSL as the signal for BmaR1-BmaI1 quorum sensing and show that octanoyl-HSL and BmaR1 activate
bmaI1
transcription.
Mycobacterial Nonhomologous End Joining Mediates Mutagenic Repair of Chromosomal Double-Strand DNA Breaks
Authors: Stephanou Nicolas C. , Gao Feng , Bongiorno Paola , Ehrt Sabine , Schnappinger Dirk , Shuman Stewart , Glickman Michael S.
Published: May 2007

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient
Mycobacterium smegmatis
strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An
M. smegmatis
Δ
recA
Δ
ku
double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.
Functional Comparison of the Two
Bacillus anthracis
Glutamate Racemases
Authors: Dodd Dylan , Reese Joseph G. , Louer Craig R. , Ballard Jimmy D. , Spies M. Ashley , Blanke Steven R.
Published: May 2007

Glutamate racemase activity in
Bacillus anthracis
is of significant interest with respect to chemotherapeutic drug design, because
l
-glutamate stereoisomerization to
d
-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of
B. anthracis
predicts two genes encoding glutamate racemases,
racE1
and
racE2
. To evaluate whether
racE1
and
racE2
encode functional glutamate racemases, we cloned and expressed
racE1
and
racE2
in
Escherichia coli
. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of
l
-glutamate and
d
-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of
l
-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that
racE1
and
racE2
encode functional glutamate racemases with similar, but not identical, active site features.
Generation of a Membrane Potential by
Lactococcus lactis
through Aerobic Electron Transport
Authors: Brooijmans R. J. W. , Poolman B. , Schuurman-Wolters G. K. , de Vos W. M. , Hugenholtz J.
Published: May 2007

Lactococcus lactis
, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in
L. lactis
under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3′,3′-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Δψ). Wild-type cells, grown aerobically in the presence of heme, generated a Δψ even in the presence of the F
1
-F
o
ATPase inhibitor
N
,
N
′-dicyclohexylcarbodiimide, while a cytochrome
bd
-negative mutant strain (CydAΔ) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydAΔ cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the
L. lactis
ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.
Effect of a
recD
Mutation on DNA Damage Resistance and Transformation in
Deinococcus radiodurans
Authors: Servinsky Matthew D. , Julin Douglas A.
Published: May 2007

The bacterium
Deinococcus radiodurans
is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in
Escherichia coli
and many other bacteria. The
D. radiodurans
genome sequence indicates that the organism lacks
recB
and
recC
genes, but there is a gene encoding a protein with significant similarity to the RecD protein of
E. coli
and other bacteria. We have generated
D. radiodurans
strains with a disruption or deletion of the
recD
gene. The
recD
mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The
recD
mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the
D. radiodurans
RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.
Distinct Centromere-LikeparSSites on the Two Chromosomes ofVibriospp
Authors: Yamaichi Yoshiharu , Fogel Michael A. , McLeod Sarah M. , Hui Monica P. , Waldor Matthew K.
Published: May 2007

Vibrio cholerae, the cause of cholera, has two circular chromosomes. TheparABgenes on eachV. choleraechromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI)parABgenes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII)parABgenes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified theparSsites on bothV. choleraechromosomes. We found three clustered origin-proximal ParB1 bindingparS1sites on ChrI. Deletion of these threeparS1sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in aparA1mutant, mislocalization of the ChrI origin region in theparS1mutant did not compromiseV. choleraegrowth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 bindingparS2sites, which differed in sequence fromparS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognateparSsequence.parABS2appears to form a functional partitioning system, as we found thatparABS2was sufficient to stabilize an ordinarily unstable plasmid inEscherichia coli. MostparS2sites were located within 70 kb of the ChrII origin of replication, but oneparS2site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution ofparS1andparS2sites was entirely chromosome specific.
Transcriptional Regulation of the
virR
Operon of the Intracellular Pathogen
Rhodococcus equi
Authors: Byrne Gavin A. , Russell Dean A. , Chen Xiaoxiao , Meijer Wim G.
Published: May 2007

The
virR
operon, located on the virulence plasmid of the intracellular pathogen
Rhodococcus equi
, contains five genes, two of which (
virR
and
orf8
) encode transcriptional regulators. The first gene of the operon (
virR
), encoding a LysR-type transcriptional regulator, is transcribed at a constitutive low level, whereas the four downstream genes are induced by low pH and high growth temperature. Differential regulation of the
virR
operon genes could not be explained by differential mRNA stability, as there were no major differences in mRNA half-lives of the transcripts representing each of the five genes within the
virR
operon. Transcription of
virR
is driven by the P

virR

promoter, with a transcription start site 53 bp upstream of the
virR
initiation codon. The four genes downstream of
virR
are transcribed from P

virR

and from a second promoter, P

orf5

, located 585 bp downstream of the
virR
initiation codon. VirR binds to a site overlapping the initiation codon of
virR
, resulting in negative autoregulation of the
virR
gene, explaining its low constitutive transcription level. The P

orf5

promoter is induced by high temperature and low pH, thus explaining the observed differential gene expression of the
virR
operon. VirR has a positive effect on P

orf5

activity, whereas the response regulator encoded by
orf8
is not involved in regulating transcription of the
virR
operon. The P

virR

promoter is strikingly similar to those recognized by the principal sigma factors of
Streptomyces
and
Mycobacterium
, whereas the P

orf5

promoter does not share sequence similarity with P

virR

. This suggests that P

orf5

is recognized by an alternative sigma factor.
NrdR Controls Differential Expression of the
Escherichia coli
Ribonucleotide Reductase Genes
Authors: Torrents Eduard , Grinberg Inna , Gorovitz-Harris Batia , Lundström Hanna , Borovok Ilya , Aharonowitz Yair , Sjöberg Britt-Marie , Cohen Gerald
Published: May 2007

Escherichia coli
possesses class Ia, class Ib, and class III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic class Ia
nrdAB
RNR genes are well expressed, whereas the aerobic class Ib
nrdEF
RNR genes are poorly expressed. The class III RNR is normally expressed under microaerophilic and anaerobic conditions. In this paper, we show that the
E. coli
YbaD protein differentially regulates the expression of the three sets of genes. YbaD is a homolog of the
Streptomyces
NrdR protein. It is not essential for growth and has been renamed NrdR. Previously,
Streptomyces
NrdR was shown to transcriptionally regulate RNR genes by binding to specific 16-bp sequence motifs, NrdR boxes, located in the regulatory regions of its RNR operons. All three
E. coli
RNR operons contain two such NrdR box motifs positioned in their regulatory regions. The NrdR boxes are located near to or overlap with the promoter elements. DNA binding experiments showed that NrdR binds to each of the upstream regulatory regions. We constructed deletions in
nrdR
(
ybaD
) and showed that they caused high-level induction of transcription of the class Ib RNR genes but had a much smaller effect on induction of transcription of the class Ia and class III RNR genes. We propose a model for differential regulation of the RNR genes based on binding of NrdR to the regulatory regions. The model assumes that differences in the positions of the NrdR binding sites, and in the sequences of the motifs themselves, determine the extent to which NrdR represses the transcription of each RNR operon.
CcpA-Dependent and -Independent Control of Beta-Galactosidase Expression in
Streptococcus pneumoniae
Occurs via Regulation of an Upstream Phosphotransferase System-Encoding Operon
Authors: Kaufman Greer E. , Yother Janet
Published: May 2007

A spontaneous mutant of
Streptococcus pneumoniae
strain D39 exhibiting elevated β-galactosidase activity was identified. We determined that the β-galactosidase activity was due to BgaA, a surface protein in
S. pneumoniae
, and that the expression of
bgaA
was regulated. Transcription analyses demonstrated expression of
bgaA
in the constitutive β-galactosidase (BgaA
C
) mutant, but not in the parent. β-Galactosidase expression was induced in the parent under specific growth conditions; however, the levels did not reach those of the BgaA
C
mutant. We localized the mutation resulting in the BgaA
C
phenotype to a region upstream of
bgaA
and in the promoter of a phosphoenolpyruvate-dependent phosphotransferase system (PTS) operon. The mutation was in a catabolite-responsive element (
cre
) and affected the binding of CcpA (catabolite control protein A), a key regulator of many carbon metabolism genes. The
pts
operon and
bgaA
were cotranscribed, and their transcription was regulated by CcpA. Deletion of
ccpA
altered β-galactosidase activity, leading to a sevenfold increase in the parent but a fivefold decrease in the BgaA
C
mutant. The resulting β-galactosidase activities were the same in the two strains, suggesting the presence of a second repressor. The presence of glucose in the growth medium resulted in
pts-bgaA
repression by both CcpA and the second repressor, with the latter being important in responding to the glucose concentration. Expression of β-galactosidase is important for
S. pneumoniae
adherence during colonization of the nasopharynx, a site normally devoid of glucose. CcpA and environmental glucose concentrations thus appear to play important roles in the regulation of a niche-specific virulence factor.
Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of theEscherichia coliADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis
Authors: Ballicora Miguel A. , Erben Esteban D. , Yazaki Terutaka , Bertolo Ana L. , Demonte Ana M. , Schmidt Jennifer R. , Aleanzi Mabel , Bejar Clarisa M. , Figueroa Carlos M. , Fusari Corina M. , Iglesias Alberto A. , Preiss Jack
Published: May 2007

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of theEscherichia coliADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for theE. colicells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102and Pro103was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.
Adaptor Function of PapF Depends on Donor Strand Exchange in P-Pilus Biogenesis of
Escherichia coli
Authors: Lee Yvonne M. , Dodson Karen W. , Hultgren Scott J.
Published: May 2007

P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis.