Α-subunit Of RNA Polymerase Research Articles

Improving the genetic stability of bacterial growth control for long-term bioproduction.

Using microorganisms for bioproduction requires the reorientation of metabolic fluxes from biomass synthesis to the production of compounds of interest. We previously engineered a synthetic growth switch in Escherichia coli based on inducible expression of theβ- and β'-subunits of RNA polymerase. Depending on the level of induction, the cells stop growing or grow at a rate close to that of the wild-type strain. This strategy has been successful in transforming growth-arrested bacteria into biofactories with a high production yield, releasing cellular resources from growth towards biosynthesis. However, high selection pressure is placed on a growth-arrested population, favoring mutations that allow cells to escape from growth control. Accordingly, we made the design of the growth switch more robust by building in genetic redundancy. More specifically, we added the rpoA gene, encoding for the α-subunit of RNA polymerase, under the control of a copy of the same inducible promoter used for expression control of ββ'. The improved growth switch is much more stable (escape frequency <10-9), while preserving the capacity to improve production yields. Moreover, after a long period of growth inhibition the population can be regenerated within a few generations. This opens up the possibility to alternate biomass accumulation and product synthesis over a longer period of time and is an additional step towards the dynamical control of bioproduction.

New evolutionary insights into RpoA, a novel quorum sensing reprograming factor in Pseudomonas aeruginosa.

Quorum-sensing (QS) coordinates the expression of virulence factors in Pseudomonas aeruginosa, an opportunistic pathogen known for causing severe infections in immunocompromised patients. QS has a master regulator, the lasR gene, but in clinical settings P. aeruginosa isolates have been found that are QS-active but LasR-null. In this study, we developed an experimental evolutionary approach to identify additional QS reprogramming determinants. We began the study with a LasR null mutant with an extra copy of mexT, a transcriptional regulator gene that is known to be able to reprogram QS in laboratory LasR-null strains. In this strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. Using this novel method, which we have named "targeted gene duplication followed by mutant screening" (TGD-MS), we identified QS-active revertants with mutations in genes other than mexT. One QS-active revertant had a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the down-regulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Our results indicate that a RpoA-dependent regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit

Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter-α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering.

Optimization of RNA Display Using GC‐Clamp Modifications to Improve Genetic Detection of RNA‐Protein Interactions

Non‐coding small regulatory RNAs (sRNAs) have an important role in bacterial stress responses. The binding of sRNAs to their target mRNAs is facilitated by RNA‐binding proteins such as Hfq or ProQ. The Berry Lab has developed a bacterial three‐hybrid (B3H) assay to detect the binding of RNA with both of these RNA chaperones in vivo, by connecting the strength of an RNA‐protein interaction to the expression of a reporter gene. The interaction between a “prey” protein fused to the α‐subunit of RNA polymerase (RNAP) and a “bait” RNA tethered upstream of a test promoter stabilizes the binding of RNAP and increases transcription of the reporter gene lacZ. Despite the promise of the B3H system and its success in detecting many high‐affinity interactions, low signal‐to‐noise for other RNA‐protein interactions currently limits the broader utility of the assay. Computational structure predictions suggested that certain RNAs of interest could misfold when hybridized with other components of the hybrid RNA construct, e.g. an MS2 hairpin (MS2hp) or an exogenous intrinsic terminator. Such misfolding would likely disrupt a bait RNA’s interaction with the prey protein. To avoid this limitation, this study aims to optimize the hybrid RNA construct to improve the breadth of detectable interactions in the B3H assay. To this end, we designed new hybrid RNA constructs with the addition of a GC‐clamp – a short insert of guanines (G) and cytosines (C) flanking a region of interest – to promote proper folding and optimal display of RNA. Several sRNAs and 5’UTRs were cloned into GC‐clamp designs and their in vivo interactions with Hfq were tested in the B3H assay. Preliminary results demonstrate the promise of a short GC‐clamp in improving the B3H signal for many sRNA‐Hfq interactions, and we are currently working to test these GC constructs with additional RNAs. Increasing the sensitivity and generalizability of the B3H assay to study bacterial RNA‐protein interactions will help shed light on the molecular mechanisms of RNA‐chaperone proteins and the important processes in bacteria they regulate, such as adaptation to stress, biofilm formation and virulence.

Articles of Significant Interest in This Issue.

The development of novel strategies allowed Camakaris et al. (e00252-21) to isolate mutants of RpoA (α-subunit of RNA polymerase) that were defective in activation of genes of the TyrR regulon and to subsequently isolate suppressors in TyrR that partially restored activation. These data demonstrated that activation involves protein-protein interaction and helped to identify both the “TyrR-specific determinant” on RpoA and the complementary “activation patch” on the TyrR N-terminal domain, both of which are surface exposed. These studies suggest a model for activation by TyrR that involves three sites on the α subunit of the C-terminal domain, the “TyrR-specific determinant,” the “265 determinant,” and the “261 determinant.”

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-Subunit of RNA Polymerase.

A novel selection was developed for mutants of the C-terminal domain of RpoA (α-CTD) altered in activation by the TyrR regulatory protein of Escherichia coli K-12. This allowed the identification of an aspartate to asparagine substitution at residue 250 (DN250) as an activation-defective (Act-) mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and the substitutions DR250, RE310, and RD310 were all shown to be defective in activation. None of these mutations caused defects in regulation of the upstream promoter (UP) element. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. The TyrR mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77, and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, and EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act-tyrR mutants were isolated in an rpoA+ strain, adding positions R77, D97, K101, D118, R119, R121, and E141 to known residues S95 and D103 and defining the activation patch on the amino-terminal domain (NTD) of TyrR. These results support a model for activation of TyrR-regulated genes where the activation patch on the TyrR NTD interacts with the TyrR-specific patch on the α-CTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants as activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene tyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore, TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence and important plant interactions.

The master quorum-sensing regulators LuxR/HapR directly interact with the alpha subunit of RNA polymerase to drive transcription activation in Vibrio harveyi and Vibrio cholerae.

In Vibrio species, quorum sensing controls gene expression for numerous group behaviors, including bioluminescence production, biofilm formation, virulence factor secretion systems, and competence. The LuxR/HapR master quorum-sensing regulators activate expression of hundreds of genes in response to changes in population densities. The mechanism of transcription activation by these TetR-type transcription factors is unknown, though LuxR DNA binding sites that lie in close proximity to the -35 region of the promoter are required for activation at some promoters. Here, we show that Vibrio harveyi LuxR directly interacts with RNA polymerase to activate transcription of the luxCDABE bioluminescence genes. LuxR interacts with RNA polymerase in vitro and in vivo and specifically interacts with both the N- and C-terminal domains of the RNA polymerase α-subunit. Amino acid substitutions in the RNAP interaction domain on LuxR decrease interactions between LuxR and the α-subunit and result in defects in transcription activation of quorum-sensing genes in vivo. The RNAP-LuxR interaction domain is conserved in Vibrio cholerae HapR and is required for activation of the HapR-regulated gene hapA. Our findings support a model in which LuxR/HapR bind proximally to RNA polymerase to drive transcription initiation at a subset of quorum-sensing genes in Vibrio species.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells.

The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the “UP element”. However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacteria isolated from a swine-manure storage pit. On the basis of the 16S rRNA, RNA polymerase α-subunit (rpoA) and 60 kDa chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA gene sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C(16:0), C(16:1) ω7c, C(16:1) ω7c, and C(18:1) ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) PPC27A = CCUG 61369; PPC38 = CCUG 61261 [corrected] and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) PPC15 = CCUG 61368; PPC107 = CCUG 61372 [corrected] are proposed for these hitherto undescribed species.

Screening for the Interacting Partners of the Proteins MamK &amp; MamJ by Two-Hybrid Genomic DNA Library of Magnetospirillum magneticum AMB-1

Magnetotactic bacteria are a group of prokaryotes capable of sensing and navigating along the earth's magnetic field. The linear alignment of magnetosomes, which acts as a compass needle for orientation, is dependent on the proteins MamJ (amb0964) & MamK (amb0965). We constructed Magnetospirillum magneticum AMB-1 two-hybrid DNA libraries by fusing the random genomic fragments of AMB-1 to the N-terminal domain of the α-subunit of RNA polymerase in vector pTRG and used as preys. The genes mamJ & mamK were cloned in frame with the λ repressor protein (λ cI) in vector pBT and used as baits for screening the binding partners. After preliminary screening, we further confirmed the candidate interactions between selected protein pairs. The results showed that there were relatively strong interactions between MamK versus Amb3498 (flagella motor switch protein fliM), versus Amb0854 MCPs (signal domain of methyl-accepting chemotaxis protein) and versus Amb3568 (GGDEF domain-containing protein), respectively. MamJ versus Amb1722 (hypothetical protein), MamJ versus MamK, and MamK versus Amb1807 (cation transport ATPase) exhibited low level of interaction. Although the TPR repeat protein MamA (amb0971) showed no interaction with either MamJ or MamK, the TPR repeat protein Amb0024 with more motif sequences exhibited relatively strong interaction with MamK. Among the identified proteins, all categorized as signal transduction-related displayed interaction only with MamK and without MamJ, suggesting that magnetotaxis via MamK in Magnetospirillum magneticum AMB-1 might be somehow concerned with the widely accepted chemotaxis mechanism in bacteria.

The HARE-HTH and associated domains

Human ASXL proteins, orthologs of Drosophila Additional Sex combs, have been implicated in conjunction with TET2 as a major target for mutations and translocations leading to a wide range of myeloid leukemias, related myelodysplastic conditions (ASXL1 and ASXL2) and the Bohring-Opitz syndrome, a developmental disorder (ASXL1). Using sensitive sequence and structure comparison methods, we show that most animal ASXL proteins contain a novel N-terminal domain that is also found in several other eukaryotic chromatin proteins, diverse restriction endonucleases and DNA glycosylases, the RNA polymerase delta subunit of Gram-positive bacteria and certain bacterial proteins that combine features of the RNA polymerase α-subunit and sigma factors. This domain adopts the winged helix-turn-helix fold and is predicted to bind DNA. Based on its domain architectural contexts, we present evidence that this domain might play an important role, both in eukaryotes and bacteria, in the recruitment of diverse effector activities, including the Polycomb repressive complexes, to DNA, depending on the state of epigenetic modifications such as 5-methylcytosine and its oxidized derivatives. In other eukaryotic chromatin proteins, this predicted DNA-binding domain is fused to a region with three conserved motifs that are also found in diverse eukaryotic chromatin proteins, such as the animal BAZ/WAL proteins, plant HB1 and MBD9, yeast Itc1p and Ioc3, RSF1, CECR2 and NURF1. Based on the crystal structure of Ioc3, we establish that these motifs in conjunction with the DDT motif constitute a structural determinant that is central to nucleosomal repositioning by the ISWI clade of SWI2/SNF2 ATPases. We also show that the central domain of the ASXL proteins (ASXH domain) is conserved outside of animals in fungi and plants, where it is combined with other domains, suggesting that it might be an ancient module mediating interactions between chromatin-linked protein complexes and transcription factors via its conserved LXLLL motif. We present evidence that the C-terminal PHD finger of ASXL protein has certain peculiar structural modifications that might allow it to recognize internal modified lysines other than those from the N terminus of histone H3, making it the mediator of previously unexpected interactions in chromatin.

Cyclic AMP Receptor Protein Regulates cspE , an Early Cold-Inducible Gene, in Escherichia coli

cspE, a member of the cspA family of cold shock proteins in Escherichia coli, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulates cspE in E. coli. Sequence analysis of the cspE upstream region revealed a putative CRP target site centered at -61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for P(cspE) activation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-P(cspE) interaction. Based on the promoter architecture, we classified P(cspE) as a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in P(cspE) transcription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in P(cspE) transcription. In addition, the deletion of crp led to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.

A Strategy for Antagonizing Quorum Sensing

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ∼60 Å, twice the ∼30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multidomain proteins.

H-NS Binding and Repression of the ctx Promoter in Vibrio cholerae

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the -35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the α-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.

Identification of Multiple Substrates of the StkP Ser/Thr Protein Kinase in Streptococcus pneumoniae

Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by two-component systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotic-type Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mn-dependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the alpha-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein- and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.

The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.

Promoter Strength Properties of the Complete Sigma E Regulon of Escherichia coli and Salmonella enterica

The sigma(E)-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. sigma(E) is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the sigma(E) regulon is driven from a suite of approximately 60 sigma(E)-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the sigma(E) promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (-35 to -65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the alpha-subunit of RNA polymerase; (ii) ppGpp generally activates sigma(E) promoters, and (iii) sigma(E) promoters are responsive to changing sigma(E) holoenzyme levels under physiological conditions, reinforcing the idea that the sigma(E) regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

Enterococcus thailandicus sp. nov., isolated from fermented sausage ('mum') in Thailand

A Gram-positive and catalase-negative coccus that formed chains, designated strain FP48-3(T), isolated from fermented sausage ('mum'), was studied systematically. Strain FP48-3(T) was facultatively anaerobic and produced l-lactic acid from glucose. Straight-chain fatty acids C(18 : 1) and C(16 : 0) were the dominant components. The DNA G+C content of strain FP48-3(T) was 37.9 mol%. On the basis of 16S rRNA and RNA polymerase alpha-subunit (rpoA) gene sequence analysis, strain FP48-3(T) was closely related to Enterococcus hirae LMG 6399(T), Enterococcus durans LMG 10746(T) and Enterococcus faecium LMG 11423(T), with 99.3-99.6 and 95.1-96.9 % sequence similarities, respectively. Strain FP48-3(T) could be clearly distinguished from E. hirae LMG 6399(T), E. durans LMG 10746(T) and E. faecium LMG 11423(T) by low DNA-DNA relatedness (< or =14 %) and phenotypic characteristics. Therefore, this strain represents a novel species of the genus Enterococcus, for which the name Enterococcus thailandicus sp. nov. is proposed. The type strain is FP48-3(T) (=KCTC 13134(T)=NBRC 101867(T)=NRIC 0107(T)=TISTR 933(T)=PCU 282(T)).