Putative Two-component System Research Articles

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa.

The identification of targets whose inactivation increases the activity of antibiotics helps to fight antibiotic resistance. Previous work showed that a transposon-insertion mutant in the gene PA14_27940 increases Pseudomonas aeruginosa susceptibility to aminoglycosides. Since polar effects may affect the phenotype, in the present work, we generated an in-frame PA14_27940 deletion mutant. A PA14_27940 deletion increased the susceptibility to aminoglycosides, tetracycline, tigecycline, erythromycin and fosfomycin. Excepting fosfomycin, the other antibiotics are inducers of the MexXY efflux pump. MexXY induction is required for P. aeruginosa resistance to these antibiotics, which is post-transcriptionally regulated by the anti-repressor ArmZ. Although mexXY is inducible by tobramycin in ΔPA14_27940, the induction level is lower than in the parental PA14 strain. Additionally, armZ is induced by tobramycin in PA14 and not in ΔPA14_27940, supporting that ΔPA14_27940 presents an ArmZ-mediated defect in mexXY induction. For its part, hypersusceptibility to fosfomycin may be due to a reduced expression of nagZ and agmK, which encode enzymes of the peptidoglycan recycling pathway. ΔPA14_27940 also presents defects in motility, an element with relevance in P. aeruginosa's virulence. Overall, our results support that PA14_27940 is a good target for the search of adjuvants that will increase the activity of antibiotics and reduce the virulence of P. aeruginosa.

The YmgB-SpoT interaction triggers the stringent response in Escherichia coli

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent from its proposed role in the two component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Role of the Solute-Binding Protein CuaD in the Signaling and Regulating Pathway of Cellobiose and Cellulose Utilization in Ruminiclostridium cellulolyticum.

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for cellobiose and cellulose utilization. Here, we show that a second SBP (CuaD), whose gene is adjacent to two genes encoding a putative two-component regulation system (CuaSR), forms a three-component system with CuaS and CuaR. Studies of mutant and recombinant strains of R. cellulolyticum have indicated that cuaD is important for the growth of strains on cellobiose and cellulose. Furthermore, the results of our RT-qPCR experiments suggest that both the three (CuaDSR)- and the two (CuaSR)-component systems are able to perceive the cellobiose signal. However, the strain producing the three-component system is more efficient in its cellobiose and cellulose utilization. As CuaD binds to CuaS, we propose an in-silico model of the complex made up of two extracellular domains of CuaS and two of CuaD. CuaD allows microorganisms to detect very low concentrations of cellobiose due to its high affinity and specificity for this disaccharide, and together with CuaSR, it triggers the expression of the cuaABC-cbpA genes involved in cellodextrins uptake.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

Genome-wide characterization of two-component system (TCS) genes in melon (Cucumis melo L.)

To better understand cytokinin signaling in melon (Cucumis melo L.), one of the most important fruit crops in the Cucurbitaceae family, we identified and characterized melon two-component system (TCS) genes in this study. The results showed that there were 51 genes encoding putative TCS proteins in melon, and these TCS genes were classified into 3 subgroups, with 17 HK(L)s (histidine kinase/histidine-kinase like; 9 HKs and 8 HKLs), 9 HPs (histidine phosphotransfer proteins; 6 authentic and 3 pseudo), and 25 RRs (response regulators; 8 Type-A, 11 Type-B and 6 pseudo). The identity values of these cytokinin signaling proteins were revealed by analyzing their conserved motifs, domains and amino acid sequences. By analyzing TCS genes in different plant species, we found that melon HK(L)s, HPs and RRs had closer phylogenetic relationships with cucumber genes than with the genes of other plants, and the expansion of melon cytokinin signaling genes might be attributed to segmental duplication events. Analysis of the putative promoter regions (2-kb upstream regions of the start codon) revealed the enrichment of stress- and hormone-response cis-elements. The involvement of these putative TCS genes in melon cytokinin signaling was further supported by qRT-PCR data.

Thiol-based redox sensing in the methyltransferase associated sensor kinase RdmS in Methanosarcina acetivorans.

Organisms have evolved signal transduction systems to quickly adapt their lifestyle to internal and environmental changes. While protein kinases and two-component systems are widely distributed in Bacteria, they are also found in Archaea but are less diversified and abundant. In this work, we analysed the function of the kinase RdmS and its role in a putative two-component system in the methanogenic archaeon Methanosarcina acetivorans. RdmS is encoded upstream of the regulator MsrF, which activates the expression of the corrinoid/methyltransferase fusion protein MtsD. In contrast to a typical bacterial histidine kinase, RdmS lacks a membrane domain and the conserved histidine residue for phosphorylation, indicating a different mechanism of signal transduction in comparison to bacterial counterparts. RdmS covalently binds a heme cofactor and is thereby able to bind small molecules like CO and dimethyl sulfide. Interestingly, RdmS possesses a redox-dependent autophosphorylation activity, which, however, is independent of the bound heme cofactor. In fact, our experimental data suggest a thiol-based redox sensing mechanism by RdmS. Moreover, we were able to show that RdmS interacts with the regulator protein MsrF. From these data, we conclude RdmS to be a thiol-based kinase sensing redox changes and forming an archaeal multicomponent system with the regulators MsrG/F/C.

Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray.

The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated S. aureus. Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of srtA and tdk as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of MW2284 and MW2285 mutants compared to wild-type cells demonstrated that the srtA gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate srtA and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.

The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium.

Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

Changes of enzyme activities related to oxidative stress in rice plants inoculated with random mutants of a Pseudomonas fluorescens strain able to improve plant fitness upon biotic and abiotic conditions.

The Pseudomonas fluorescens strain used in this work (Aur 6) has demonstrated its ability to improve fitness of different plant species upon biotic and abiotic stress conditions. Random mutants of this strain were constructed with the Tn5 transposon technology, and biological tests to evaluate loss of salt protection were conducted with all the mutants (104 mutants) on rice seedlings. Mutant 33 showed an evident reduction in its ability to protect plants upon salt stress challenge, whereas mutant 19 was more effective than the wild type. Enzymes related with oxidative stress were studied in both mutants and wild type. Enzyme activities were decreased with mutant 33 with regard to wild type, whereas mutant 19 did not produce important changes suggesting involvement of redox balance associated to the observed modifications in these antioxidant enzymes as one of the probable mechanisms used by these strains. Data of malondialdehyde (MDA) were consistent with this fact. Mutants also affected accumulation of proline, the most common osmolyte in plants. A second experiment to evaluate the ability of both mutants and wild type to stimulate growth on tomato plants was conducted, as this feature was previously demonstrated by wild type. Similar results were obtained in growth of both species, suggesting that mutations of both mutants are related with the capacities of the wild type to stimulate growth. To reveal mutated genes, both mutants were mapped. Three mutated genes were found in mutant 33. A gene related with a general secretion pathway protein D, a gene related with a putative two-component system sensor kinase (ColS), and a gene related with flagellar motor switch protein (FliG). In mutant 19, two mutated genes were found. One gene related with heavy metal efflux pump Czca family, and other gene of 16s rRNA.

Sequences of Landomycin E and Carotenoid Biosynthetic Gene Clusters, and Molecular Structure of Transcriptional Regulator of Streptomyces globisporus 1912

Streptomyces globisporus 1912 and its derivatives 1912-2 and 1912-4Crt are the producers of the landomycin E, carotenoids and the regulator of antibiotics biosynthesis and morphogenesis of streptomycetes. The genome DNA of two mutant strains, 1912-2, the more effective producer of the landomycin E and the regulator, and 1912-4Crt, the producer of beta-carotene and lycopene, was sequenced by Illumina. Comparative analysis of the DNA sequences using GenBank data allowed localization of 36 landomycin E biosynthetic genes lnd of S. globisporus 1912 in one cluster. Twenty of these lnd genes have been sequenced for the first time. The new regulatory responce gene lndRR and the sensor kinase gene lndY1 were proposed as the members of the putative two-component system. High identity (94 – 95 %) was determined for the lnd genes of the 1912 strain and those of the metagenomic clone AZ97. Seven carotenoid biosynthetic genes crt of the strain 1912-4Crt were sequenced and localized in one cluster consisting of two convergent operons from 4 and 3 crt genes. High homology (93 %) of the crt gene clusters of S. globisporus 1912 and S. griseus IFO 13350 was shown. Two non-punctual repeats (NPRs) of 21 bp were identified in the sequence of crtY gene coding lycopene cyclase. It was shown that the deletion of 117 bp including the sequence between NPRs of 96 bp and one NPR from 5`-side activated the crt gene cluster and increased the production of beta carotene (6.91 mg/l) and lycopene (3.24 mg/l) by the strain 1912-4Crt. Deletion of 86 bp was revealed in the regulatory gene lndRR resulting in the deficiency of landomycin E production in the strain 1912-4Crt. The DNA sequences of crt and lnd genes of S. globisporus 1912 were submitted to the NCBI database with accession numbers KM349312 and KJ645792, respectively. S. globisporus 1912 produced a low-molecular-weight compound that, like A-factor, restored the landomycin E and streptomycin biosynthesis and sporulation of the defective mutants S. globisporus 1912-B2 and S. griseus 1439, respectively. The compound was purified by thin layer chromatography and HPLC. It had an absorption maximum at λmax= 245 nm and a molecular mass m/z 244. On the basis of NMR spectroscopy the chemical structure of the transcriptional regulator was elucidated as the new (L)-N-methylphenylalanyl-dehydrobutyrine diketopiperazine.

Multilocus Sequence Typing reveals high variability among Clavibacter michiganensis subsp. michiganensis strains affecting tomato crops in Uruguay

Bacterial canker caused by Clavibacter michiganensis subspecies michiganensis (Cmm) has been a threat to tomato production in Uruguay for several years. In this study, 39 Cmm strains were collected from several outbreaks and production areas in Uruguay, and were identified by molecular assays and pathogenicity tests on a susceptible cultivar of tomato. In addition, a TaqMan assay targeting a putative two-component system sensor kinase gene demonstrated good specificity with all strains tested and gave no false negative results. The first epidemiological study of Cmm in this country was carried out in order to elucidate the origin of outbreaks and sources of infection and dissemination of the pathogen. Strains from Uruguay showed high genetic diversity based on a Multi Locus Sequence Typing analysis of five housekeeping genes. This approach revealed 36 sequence types (STs) within a worldwide collection of 108 Cmm strains. Ten STs correspond to strains solely isolated in Uruguay, including eight novel STs for the subspecies michiganensis. This high diversity reflects the introduction of new strains from different origins that most probably results from seed importation. This study provides relevant information about the distribution and origin of strains causing bacterial canker in Uruguay and will pave the way for the establishment of preventive measures to control the disease.

Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K(+) homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K(+) uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K(+) (Kdp). The contributions of each of these K(+) transport systems to cellular K(+) homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K(+) uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K(+) uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K(+) depletion. Correspondingly, Kdp-mediated K(+) uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K(+) required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K(+) concentration under conditions of limited K(+) in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K(+) availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K(+) levels under fluctuating environmental conditions.

Quorum sensing in group A Streptococcus.

Quorum sensing (QS) is a widespread phenomenon in the microbial world that has important implications in the coordination of population-wide responses in several bacterial pathogens. In Group A Streptococcus (GAS), many questions surrounding QS systems remain to be solved pertaining to their function and their contribution to the GAS lifestyle in the host. The QS systems of GAS described to date can be categorized into four groups: regulator gene of glucosyltransferase (Rgg), Sil, lantibiotic systems, and LuxS/AI-2. The Rgg family of proteins, a conserved group of transcription factors that modify their activity in response to signaling peptides, has been shown to regulate genes involved in virulence, biofilm formation and competence. The sil locus, whose expression is regulated by the activity of signaling peptides and a putative two-component system (TCS), has been implicated on regulating genes involved with invasive disease in GAS isolates. Lantibiotic regulatory systems are involved in the production of bacteriocins and their autoregulation, and some of these genes have been shown to target both bacterial organisms as well as processes of survival inside the infected host. Finally AI-2 (dihydroxy pentanedione, DPD), synthesized by the LuxS enzyme in several bacteria including GAS, has been proposed to be a universal bacterial communication molecule. In this review we discuss the mechanisms of these four systems, the putative functions of their targets, and pose critical questions for future studies.

An extracytoplasmic function sigma factor, σ(25), differentially regulates avermectin and oligomycin biosynthesis in Streptomyces avermitilis.

σ(25) is an extracytoplasmic function (ECF) σ factor in the bacterium Streptomyces avermitilis that plays a differential regulatory role in avermectin and oligomycin biosynthesis. Gene deletion, complementation, and overexpression experiments showed that σ(25) inhibited avermectin production but promoted oligomycin production. σ(25) indirectly inhibited avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activated oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. The divergently transcribed genes smrAB are located upstream of sig25 and encode a putative two-component system (TCS). σ(25) was found to initiate its own transcription, and its expression was directly activated by SmrA. The precise SmrA-binding sites in the region upstream of sig25 were determined by DNase I footprinting assays and identified two direct repeat sequences CTGTGA-n5-CTGTGA, suggesting that SmrA regulates sig25 transcription by binding to these direct repeats. The deletion of smrAB had the similar effect on avermectin and oligomycin A production to the deletion of sig25, indicating that σ(25) and SmrAB function similarly in the regulation of antibiotic production. These findings helpfully clarify the regulation of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in S. avermitilis.

Identification of a putative cognate sensor kinase for the two‐component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris

The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.

A Two-Component System (XydS/R) Controls the Expression of Genes Encoding CBM6-Containing Proteins in Response to Straw in Clostridium cellulolyticum

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.

A protein secretion system linked to bacteroidete gliding motility and pathogenesis

Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

The Novel Genes emmABC Are Associated with Exopolysaccharide Production, Motility, Stress Adaptation, and Symbiosis in Sinorhizobium meliloti

The nitrogen-fixing symbiont Sinorhizobium meliloti senses and responds to constantly changing environmental conditions as it makes its way through the soil in search of its leguminous plant host, Medicago sativa (alfalfa). As a result, this bacterium regulates various aspects of its physiology in order to respond appropriately to stress, starvation, and competition. For example, exopolysaccharide production, which has been shown to play an important role in the ability of S. meliloti to successfully invade its host, also helps the bacterium withstand osmotic changes and other environmental stresses. In an effort to further elucidate the intricate regulation of this important cell component, we set out to identify genetic factors that may affect its production. Here we characterize novel genes that encode a small protein (EmmA) and a putative two-component system (EmmB-EmmC). A mutation in any of these genes leads to increased production of the symbiotically important exopolysaccharide succinoglycan. In addition, emm mutants display membrane-associated defects, are nonmotile, and are unable to form an optimal symbiosis with alfalfa, suggesting that these novel genes may play a greater role in the overall fitness of S. meliloti both during the free-living stage and in its association with its host.

The two-component system BfrAB regulates expression of ABC transporters in Streptococcus gordonii and Streptococcus sanguinis

The putative two-component system BfrAB is involved in Streptococcus gordonii biofilm development. Here, we provide evidence that BfrAB regulates the expression of bfrCD and bfrEFG, which encode two ATP-binding cassette (ABC) transporters, and bfrH, which encodes a CAAX amino-terminal protease family protein. BfrC and BfrE are ATP-binding proteins, and BfrD, BfrF and BfrG are homologous membrane-spanning polypeptides. Similarly, BfrABss, the BfrAB homologous system in Streptococcus sanguinis, controls the expression of two bfrCD-homologous operons (bfrCDss and bfrXYss), a bfrH-homologous gene (bfrH1ss) and another CAAX amino-terminal protease family protein gene (bfrH2ss). Furthermore, we demonstrate that the purified BfrA DNA-binding domain from S. gordonii binds to the promoter regions of bfrCD, bfrEFG, bfrH, bfrCDss, bfrXYss and bfrH1ss in vitro. Finally, we show that the BfrA DNA-binding domain recognizes a conserved DNA motif with a consensus sequence of TTTCTTTAGAAATATTTTAGAATT. These data suggest, therefore, that S. gordonii BfrAB controls biofilm formation by regulating multiple ABC-transporter systems.