Filifactor alocis enhances survival of Porphyromonas gingivalis W83 in response to H2 O2 -induced stress.

A dysbiotic microbial community whose members have specific/synergistic functions that are modulated by environmental conditions, can disturb homeostasis in the subgingival space leading to destructive inflammation, plays a role in the progression of periodontitis. Filifactor alocis, a gram-positive, anaerobic bacterium, is a newly recognized microbe that shows a strong correlation with periodontal disease. Our previous observations suggested F. alocis to be more resistant to oxidative stress compared to Porphyromonas gingivalis. The objective of this study is to further determine if F. alocis, because of its increased resistance to oxidative stress, can affect the survival of other 'established' periodontal pathogens under environmental stress conditions typical of the periodontal pocket. Here, we have shown that via their interaction, F. alocis protects P. gingivalis W83 under H2 O2 -induced oxidative stress conditions. Transcriptional profiling of the interaction of F. alocis and P. gingivalis in the presence of H2 O2 -induced stress revealed the modulation of several genes, including those with ABC transporter and other cellular functions. The ABC transporter operon (PG0682-PG0685) of P. gingivalis was not significant to its enhanced survival when cocultured with F. alocis under H2 O2 -induced oxidative stress. In F. alocis, one of the most highly up-regulated operons (FA0894-FA0897) is predicted to encode a putative manganese ABC transporter, which in other bacteria can play an essential role in oxidative stress protection. Collectively, the results may indicate that F. alocis could likely stabilize the microbial community in the inflammatory microenvironment of the periodontal pocket by reducing the oxidative environment. This strategy could be vital to the survival of other pathogens, such as P. gingivalis, and its ability to adapt and persist in the periodontal pocket.

The involvement of CdhR in Porphyromonas gingivalis during nitric oxide stress.

Porphyromonas gingivalis, the causative agent of adult periodontitis, must gain resistance to frequent oxidative and nitric oxide (NO) stress attacks from immune cells in the periodontal pocket to survive. Previously, we found that, in the wild-type and under NO stress, the expression of PG1237 (CdhR), the gene encoding for a putative LuxR transcriptional regulator previously called community development and hemin regulator (CdhR), was upregulated 7.7-fold, and its adjacent gene PG1236 11.9-fold. Isogenic mutants P. gingivalis FLL457 (ΔCdhR::ermF), FLL458 (ΔPG1236::ermF), and FLL459 (ΔPG1236-CdhR::ermF) were made by allelic exchange mutagenesis to determine the involvement of these genes in P. gingivalis W83 NO stress resistance. The mutants were black pigmented and ß hemolytic and their gingipain activities varied with strains. FLL457 and FLL459 mutants were more sensitive to NO compared to the wild type, and complementation restored NO sensitivity to that of the wild type. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were upregulated and over 1% of the genes downregulated under NO stress conditions compared to the wild type. Transcriptome analysis of FLL458 and FLL459 under NO stress showed differences in their modulation patterns. Some similarities were also noticed between all mutants. The PG1236-CdhR gene cluster revealed increased expression under NO stress and may be part of the same transcriptional unit. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495. Taken together, the data indicate that CdhR may play a role in NO stress resistance and be involved in a regulatory network in P. gingivalis.

Local and Systemic Effects of Porphyromonas gingivalis Infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase.

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Role of the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance.

In the periodontal pocket, there is a direct correlation between environmental conditions, the dynamic oral microbial flora, and disease. The relative abundance of several newly recognized microbial species in the oral microenvironment has raised questions on their impact on disease development. One such organism, Filifactor alocis, is significant to the pathogenic biofilm structure. Moreover, its pathogenic characteristics are highlighted by its ability to survive in the oxidative-stress microenvironment of the periodontal pocket and alter the microbial community dynamics. There is a gap in our understanding of its mechanism(s) of oxidative stress resistance and impact on pathogenicity. Several proteins, including HMPRFF0389-00519 (FA519), were observed in high abundance in F. alocis during coinfection of epithelial cells with Porphyromonas gingivalis W83. Bioinformatics analysis shows that FA519 contains a "Cys-X-X-Cys zinc ribbon domain" which could be involved in DNA binding and oxidative stress resistance. We have characterized FA519 to elucidate its roles in the oxidative stress resistance and virulence of F. alocis. Compared to the wild-type strain, the F. alocis isogenic gene deletion mutant, FLL1013 (ΔFA519::ermF), showed significantly reduced sensitivity to hydrogen peroxide and nitric oxide-induced stress. The ability to form biofilm and adhere to and invade gingival epithelial cells was also reduced in the isogenic mutant. The recombinant FA519 protein was shown to protect DNA from Fenton-mediated damage with an intrinsic ability to reduce hydrogen peroxide and disulfide bonds. Collectively, these results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis. IMPORTANCE Filifactor alocis is an emerging member of the periodontal community and is now proposed to be a diagnostic indicator of periodontal disease. However, due to the lack of genetic tools available to study this organism, not much is known about its virulence attributes. The mechanism(s) of oxidative stress resistance in F. alocis is unknown. Therefore, identifying the adaptive mechanisms utilized by F. alocis to survive in the oxidative stress environment of the periodontal pocket would lead to understanding its virulence regulation, which could help develop novel therapeutic treatments to combat the effects of periodontal disease. This study is focused on the characterization of FA519, a hypothetical protein in F. alocis, as a multifunctional protein that plays an important role in the reactive oxygen species-detoxification pathway. Collectively, our results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis.

PG1659 functions as anti-sigma factor to extracytoplasmic function sigma factor RpoE in Porphyromonas gingivalis W83.

Anti-sigma factors play a critical role in regulating the expression of sigma factors in response to environmental stress signals. PG1659 is cotranscribed with an upstream gene PG1660 (rpoE), which encodes for a sigma factor that plays an important role in oxidative stress resistance and the virulence regulatory network of P. gingivalis. PG1659, which is annotated as a hypothetical gene, is evaluated in this study. PG1659, composed of 130 amino acids, is predicted to be transmembrane protein with a single calcium (Ca2+ ) binding site. In P. gingivalis FLL358 (ΔPG1659::ermF), the rpoE gene was highly upregulated compared to the wild-type W83 strain. RpoE-induced genes were also upregulated in the PG1659-defective isogenic mutant. Both protein-protein pull-down and bacterial two-hybrid assays revealed that the PG1659 protein could interact with/bind RpoE. The N-terminal domain of PG1659, representing the cytoplasmic fragment of the protein, is critical for interaction with RpoE. In the presence of PG1659, the initiation of transcription by the RpoE sigma factor was inhibited. Taken together, our data suggest that PG1659 is an anti-sigma factor which plays an important regulatory role in the modulation of the sigma factor RpoE in P. gingivalis.

Correction: In Porphyromonas gingivalis VimF Is Involved in Gingipain Maturation through the Transfer of Galactose.

[This corrects the article DOI: 10.1371/journal.pone.0063367.].

Role of Acetyltransferase PG1842 in Gingipain Biogenesis in Porphyromonas gingivalis.

Porphyromonas gingivalis, the major etiologic agent in adult periodontitis, produces large amounts of proteases that are important for its survival and pathogenesis. The activation/maturation of gingipains, the major proteases, in P. gingivalis involves a complex network of processes which are not yet fully understood. VimA, a putative acetyltransferase and virulence-modulating protein in P. gingivalis, is known to be involved in gingipain biogenesis. P. gingivalis FLL92, a vimA-defective isogenic mutant (vimA::ermF-ermAM) showed late-onset gingipain activity at stationary phase, indicating the likelihood of a complementary functional VimA homolog in that growth phase. This study aimed to identify a functional homolog(s) that may activate the gingipains in the absence of VimA at stationary phase. A bioinformatics analysis showed five putative GCN5-related N-acetyltransferases (GNAT) encoded in the P. gingivalis genome that are structurally related to VimA. Allelic exchange mutagenesis was used to make deletion mutants for these acetyltransferases in the P. gingivalisvimA-defective mutant FLL102 (ΔvimA::ermF) genetic background. One of the mutants, designated P. gingivalis FLL126 (ΔvimA-ΔPG1842), did not show any late-onset gingipain activity at stationary phase compared to that of the parent strain P. gingivalis FLL102. A Western blot analysis of stationary-phase extracellular fractions with antigingipain antibodies showed immunoreactive bands that were similar in size to those for the progingipain species present only in the ΔvimA-ΔPG1842 isogenic mutant. Both recombinant VimA and PG1842 proteins acetylated Y230, K247, and K248 residues in the pro-RgpB substrate. Collectively, these findings indicate that PG1842 may play a significant role in the activation/maturation of gingipains in P. gingivalisIMPORTANCE Gingipain proteases are key virulence factors secreted by Porphyromonas gingivalis that cause periodontal tissue damage and the degradation of the host immune system proteins. Gingipains are translated as an inactive zymogen to restrict intracellular proteolytic activity before secretion. Posttranslational processing converts the inactive proenzyme to a catalytically active protease. Gingipain biogenesis, including its secretion and activation, is a complex process which is still not fully understood. One recent study identified acetylated lysine residues in the three gingipains RgpA, RgpB, and Kgp, thus indicating a role for acetylation in gingipain biogenesis. Here, we show that the acetyltransferases VimA and PG1842 can acetylate the pro-RgpB gingipain species. These findings further indicate that acetylation is a potential mechanism in the gingipain activation/maturation pathway in P. gingivalis.

The Biofilm Community-Rebels with a Cause.

Oral Biofilms are one of the most complex and diverse ecosystem developed by successive colonization of more than 600 bacterial taxa. Development starts with the attachment of early colonizers such as Actinomyces species and oral streptococci on the acquired pellicle and tooth enamel. These bacteria not only adhere to tooth surface but also interact with each other and lay foundation for attachment of bridging colonizer such as Fusobacterium nucleatum followed by late colonizers including the red complex species: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola-the founders of periodontal disease. As the biofilm progresses from supragingival sites to subgingival sites, the environment changes from aerobic to anaerobic thus favoring the growth of mainly Gram-negative obligate anaerobes while restricting the growth of the early Gram-positive facultative aerobes. Microbes present at supragingival level are mainly related to gingivitis and root-caries whereas subgingival species advance the destruction of teeth supporting tissues and thus causing periodontitis. This review summarizes our present understanding and recent developments on the characteristic features of supra- and subgingival biofilms, interaction between different genera and species of bacteria constituting these biofilms and draws our attention to the role of some of the recently discovered members of the oral community.

Filifactor alocis--a new emerging periodontal pathogen.

Filifactor alocis, a previously unrecognized Gram-positive anaerobic rod, is now considered a new emerging pathogen that may play a significant role in periodontal disease. F. alocis' unique characteristics and variations at the molecular level that may be responsible for the functional changes required to mediate the pathogenic process are discussed.

Involvement of PG2212 zinc finger protein in the regulation of oxidative stress resistance in Porphyromonas gingivalis W83.

The adaptation of Porphyromonas gingivalis to H2O2-induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H2O2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys2His2) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H2O2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H2O2-induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis.

Proteome analysis of coinfection of epithelial cells with Filifactor alocis and Porphyromonas gingivalis shows modulation of pathogen and host regulatory pathways.

Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including Filifactor alocis, as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of F. alocis are enhanced in coculture with Porphyromonas gingivalis. We have evaluated the proteome of host cells and F. alocis during a polymicrobial infection. Coinfection of epithelial cells with F. alocis and P. gingivalis strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike F. alocis ATCC 35896, the D-62D strain expressed more proteins during coculture with P. gingivalis W83 than with P. gingivalis 33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of F. alocis may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.

In Porphyromonas gingivalis VimF is involved in gingipain maturation through the transfer of galactose.

Previously, we have reported that gingipain activity in Porphyromonas gingivalis, the major causative agent in adult periodontitis, is post-translationally regulated by the unique Vim proteins including VimF, a putative glycosyltransferase. To further characterize VimF, an isogenic mutant defective in this gene in a different P. gingivalis genetic background was evaluated. In addition, the recombinant VimF protein was used to further confirm its glycosyltransferase function. The vimF-defective mutant (FLL476) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 genetic background. While hemagglutination was not detected and autoaggregation was reduced, biofilm formation was increased in FLL476. HeLa cells incubated with P. gingivalis FLL95 and FLL476 showed a 45% decrease in their invasive capacity. Antibodies raised against the recombinant VimF protein in E. coli immunoreacted only with the deglycosylated native VimF protein from P. gingivalis. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. In the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified by mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb 1B5 antibody. Taken together, these results suggest the VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation. Moreover, galatose is vital for the growing glycan chain.

Differential response of Porphyromonas gingivalis to varying levels and duration of hydrogen peroxide-induced oxidative stress.

Porphyromonas gingivalis, an anaerobic oral pathogen implicated in adult periodontitis, can exist in an environment of oxidative stress. To evaluate its adaptation to this environment, we have assessed the response of P. gingivalis W83 to varying levels and durations of hydrogen peroxide (H(2)O(2))-induced stress. When P. gingivalis was initially exposed to a subinhibitory concentration of H(2)O(2) (0.1 mM), an adaptive response to higher concentrations could be induced. Transcriptome analysis demonstrated that oxidative stress can modulate several functional classes of genes depending on the severity and duration of the exposure. A 10 min exposure to H(2)O(2) revealed increased expression of genes involved in DNA damage and repair, while after 15 min, genes involved in protein fate, protein folding and stabilization were upregulated. Approximately 9 and 2.8% of the P. gingivalis genome displayed altered expression in response to H(2)O(2) exposure at 10 and 15 min, respectively. Substantially more genes were upregulated (109 at 10 min; 47 at 15 min) than downregulated (76 at 10 min; 11 at 15 min) by twofold or higher in response to H(2)O(2) exposure. The majority of these modulated genes were hypothetical or of unknown function. One of those genes (pg1372) with DNA-binding properties that was upregulated during prolonged oxidative stress was inactivated by allelic exchange mutagenesis. The isogenic mutant P. gingivalis FLL363 (pg1372 : : ermF) showed increased sensitivity to H(2)O(2) compared with the parent strain. Collectively, our data indicate the adaptive ability of P. gingivalis to oxidative stress and further underscore the complex nature of its resistance strategy under those conditions.

Sialidase and sialoglycoproteases can modulate virulence in Porphyromonas gingivalis.

The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 (PG0778::ermF), FLL402 (PG1724::ermF), and FLL403 (PG0352::ermF-ermAM) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism.

Identification of hydrogen peroxide production-related genes in Streptococcus sanguinis and their functional relationship with pyruvate oxidase.

Hydrogen peroxide (H(2)O(2)), an important substance produced by many members of the genus Streptococcus, plays important roles in virulence and antagonism within a microbial community such as oral biofilms. The spxB gene, which encodes pyruvate oxidase, is involved in H(2)O(2) production in many streptococcal species. However, knowledge about its regulation and relation with other genes putatively involved in the same pathway is limited. In this study, three genes--ackA, spxR and tpk--were identified as contributing to H(2)O(2) production in Streptococcus sanguinis by screening mutants for opaque colony appearance. Mutations in all three genes resulted in significant decreases in H(2)O(2) production, with 16-31% of that of the wild-type. H(2)O(2) production was restored in the complemented strains. Antagonism against Streptococcus mutans by these three S. sanguinis mutants was reduced, both on plates and in liquid cultures, indicating the critical roles of these three genes for conferring the competitive advantage of S. sanguinis. Analysis by qPCR indicated that the expression of spxB was decreased in the ackA and spxR mutants and significantly increased in the tpk mutant.

Genome-wide essential gene identification in Streptococcus sanguinis.

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.

Involvement of extracytoplasmic function sigma factors in virulence regulation in Porphyromonas gingivalis W83.

Extracytoplasmic function (ECF) sigma factors are known to play an important role in the bacterial response to various environmental stresses and can significantly modulate their pathogenic potential. In the genome of Porphyromonas gingivalis W83, six putative ECF sigma factors were identified. To further evaluate their role in this organism, a PCR-based linear transformation method was used to inactivate five ECF sigma factor genes (PG0162, PG0214, PG0985, PG1660, and PG1827) by allelic exchange mutagenesis. All five isogenic mutants formed black-pigmented colonies on blood agar. Mutants defective in PG0985, PG1660, and PG1827 genes were more sensitive to 0.25 mM of hydrogen peroxide compared with the wild-type strain. Isogenic mutants of PG0162 and PG1660 showed a 50% decrease in gingipain activity. Reverse transcription-PCR analysis showed that there was no alteration in the expression of rgpA, rgpB, and kgp gingipain genes in these mutants. Hemolytic and hemagglutination activities were decreased by more than 50% in the PG0162 mutant compared with the wild type. Taken together, these findings suggest that ECF sigma factors can modulate important virulence factors in P. gingivalis. ECF sigma factors encoded by the PG0162 and PG1660 genes might also be involved in the post-transcriptional regulation of the gingipains.

Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501.

BACKGROUND: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. RESULTS: Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-sigma54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. CONCLUSIONS: Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif-specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.

Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501.

The capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture.