Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III) oxide, Mn(IV) oxide, or electrode reduction by Geobacter sulfurreducens

10.1101/169086 ◽

2017 ◽

Cited By ~ 1

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

AbstractAt least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and −0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III)-oxide, Mn(IV)-oxide, or electrode reduction by Geobacter sulfurreducens .

10.1101/350553 ◽

2018 ◽

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

At least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c -type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and -0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an Δ omcBC background, such as extEFG , were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the Δ extABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens , depending on the available extracellular electron acceptor.

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Analysis of Cytadherence-Deficient, GapA-NegativeMycoplasma gallisepticum Strain R

Infection and Immunity ◽

10.1128/iai.68.12.6643-6649.2000 ◽

2000 ◽

Vol 68 (12) ◽

pp. 6643-6649 ◽

Cited By ~ 44

Author(s):

L. Papazisi ◽

K. E. Troy ◽

T. S. Gorton ◽

X. Liao ◽

S. J. Geary

Keyword(s):

Shuttle Vector ◽

Phenotypic Expression ◽

Mycoplasma Genitalium ◽

Transcriptional Analysis ◽

Start Codon ◽

Wild Type ◽

Gene Encoding ◽

Genes Encoding ◽

Low Passage ◽

Translational Start

ABSTRACT Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapAtranslational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapAin R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream ofgapA in the same operon and was designatedcrmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in bothMycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed thatcrmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-typegapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

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CO-Dependent H2Production by Genetically Engineered Thermococcus onnurineus NA1

Applied and Environmental Microbiology ◽

10.1128/aem.03298-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 2048-2053 ◽

Cited By ~ 57

Author(s):

Min-Sik Kim ◽

Seung Seob Bae ◽

Yun Jae Kim ◽

Tae Wan Kim ◽

Jae Kyu Lim ◽

...

Keyword(s):

Gene Cluster ◽

Type Strain ◽

Wild Type Strain ◽

Specific Activity ◽

Steel Production ◽

Transcriptional Analysis ◽

Bioreactor Culture ◽

Wild Type ◽

Steel Mill ◽

Engineered Strain

ABSTRACTHydrogenogenic CO oxidation (CO + H2O → CO2+ H2) has the potential for H2production as a clean renewable fuel.Thermococcus onnurineusNA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na+/H+antiporter and a 1.8-fold-higher specific activity for CO-dependent H2production than did the wild-type strain. The H2production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H2production rate of the engineered strain was severalfold higher than those of any other CO-dependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H2production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

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SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

Infection and Immunity ◽

10.1128/iai.01132-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2638-2645 ◽

Cited By ~ 39

Author(s):

Charlotte Michaux ◽

Maurizio Sanguinetti ◽

Fany Reffuveille ◽

Yanick Auffray ◽

Brunella Posteraro ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Galleria Mellonella ◽

Peritoneal Macrophages ◽

Bacterial Virulence ◽

Transcriptional Analysis ◽

Infection Model ◽

Wild Type ◽

Content Type ◽

Wild Type Parent

ABSTRACTPhylogenetic analysis of the crystal structure of theEnterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator inE. faecalisbiology, we dissected the genetic organization of theslyA-EF_3001 locus and constructed aslyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that theslyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

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Oligopeptide Permease A5 Modulates Vertebrate Host-Specific Adaptation of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.05234-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3407-3420 ◽

Cited By ~ 23

Author(s):

B. V. Subba Raju ◽

Maria D. Esteve-Gassent ◽

S. L. Rajasekhar Karna ◽

Christine L. Miller ◽

Tricia A. Van Laar ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Shuttle Vector ◽

Immunoblot Analysis ◽

Vertebrate Host ◽

Transcriptional Analysis ◽

Wild Type ◽

Content Type ◽

Specific Adaptation ◽

Protein Levels ◽

Oligopeptide Permease

ABSTRACTBorrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lackingoppA5was constructed in an lp25-deficient isolate ofB. burgdorferistrain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy ofbba34. Immunoblot analysis of thebba34mutant revealed a reduction in the levels of RpoS, BosR, and CsrABbwith a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, thebba34mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in thebba34mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation ofB. burgdorferito the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation ofB. burgdorferi.

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Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

Microbiology ◽

10.1099/mic.0.049387-0 ◽

2011 ◽

Vol 157 (10) ◽

pp. 2891-2903 ◽

Cited By ~ 14

Author(s):

Hyo Jung Lee ◽

Jeong Myeong Kim ◽

Se Hee Lee ◽

Minjeong Park ◽

Kangseok Lee ◽

...

Keyword(s):

Gene Cluster ◽

Transcriptional Analysis ◽

Catabolic Gene ◽

Wild Type ◽

High Sequence Identity ◽

The Third ◽

Naphthalene Degradation ◽

Knockout Mutants ◽

Gentisate Pathway ◽

High Level

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36–39 kDa, and their structures were deduced to be dimeric. The K m values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.

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Identification of Concomitant Infection with Chlamydia trachomatis IncA-Negative Mutant and Wild-Type Strains by Genomic, Transcriptional, and Biological Characterizations

Infection and Immunity ◽

10.1128/iai.00984-08 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5438-5446 ◽

Cited By ~ 27

Author(s):

Robert J. Suchland ◽

Brendan M. Jeffrey ◽

Minsheng Xia ◽

Ajay Bhatia ◽

Hencelyn G. Chu ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Genome Sequence ◽

Type Strain ◽

Wild Type Strain ◽

Genomic Sequence ◽

Genomic Structure ◽

Transcriptional Analysis ◽

Clinical Samples ◽

Wild Type

ABSTRACT Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

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The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.70.9.4968-4976.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4968-4976 ◽

Cited By ~ 65

Author(s):

Susanna Ricci ◽

Robert Janulczyk ◽

Lars Björck

Keyword(s):

Oxidative Stress ◽

Streptococcus Pyogenes ◽

Stress Responses ◽

Metal Binding ◽

Iron Homeostasis ◽

Bacterial Species ◽

Transcriptional Analysis ◽

Wild Type ◽

Allelic Replacement

ABSTRACT Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

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