Adaptation of the gut pathobiont Enterococcus faecalis to deoxycholate and taurocholate bile acids.

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.

Logic of the Escherichia coli cell cycle.

The logic of Escherichia coli's responses to environmental changes gives hope that its cell cycle will be equally well designed. During growth in a constant environment, internal signals trigger cell-cycle events such as replication initiation and cell division. Internal signals must also provide the cell with information about its present state, enabling it to coordinate the synthesis of cytoplasm, DNA and cell wall and maintain proper cell shape and composition. How the cell regulates these aspects of its growth is a fascinating--and as yet unfinished--story.

The SIGNAL experiment in BIORACK: Escherichia coli in microgravity.

Microgravity affects certain physical properties of fluids, such as convection movement and surface tension. As a consequence, cells and living organisms may exhibit different behaviour in space, which may result from differences in the immediate environment of the cell or changes in the structure of the membrane in microgravity. Two experiments to examine the effects of microgravity on cell microenvironment and signal transduction through membranes were performed using a well-characterized system with different strains of the non-pathogenic Gram-negative bacterium Escherichia coli. Our results indicate that (i) microgravity appears to reduce the lag period of a non-motile culture of E. coli, and (ii) the ompC gene, regulated by the two-component system EnvZ-OmpR, is induced as well or better in microgravity than in ground controls.

The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl-beta-D-thiogalactopyranoside exclusion and spectinomycin sensitivity.

Expression of cloned genes from isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoters is lowered when the Escherichia coli CmlA/Cmr/MdfA efflux pump is overexpressed, probably due to IPTG exclusion from the cytoplasm. The previously reported cmlA1 mutation confers a similar phenotype. cmlA1 contains an IS30 insertion upstream of cmr/mdfA, which creates a putative promoter. CmlA overproduction also causes spectinomycin hypersensitivity.

Escherichia coli metabolism in space.

Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment--weightlessness and heavy particle radiation--on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.

Screening for stabilization of proteins with a trans-translation signature in Escherichia coli selects for inactivation of the ClpXP protease.

Trans-translation is a process that adds a hydrophobic peptide tag to the C-terminus of polypeptides, which causes them to become unstable. We designed a genetic screen to identify factors involved in the degradation of trans-translated products, using the green fluorescent protein (GFP) fused to the trans-translation tag as a reporter. Two screens were devised to identify insertional mutants that stabilize such substrates. Only disruption of the clpX or clpP gene resulted in stabilization of the tagged substrates. The sspB gene product was recently shown to be a specificity-enhancing factor for the ClpXP degradation machine. In the wild-type background, targeted inactivation of the sspB gene failed to stabilize the tagged substrate. These results indicate that the ATP-dependent ClpXP protease is probably the only main component involved in the degradation of cytoplasmic trans-translated proteins in Escherichia coli that can be completely inactivated.

Regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli.

HflB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator sigma 32 and of the phage regulator lambda cll. The hflB1(Ts) allele (formerly called ftsH1) conferring temperature-sensitive growth at 42 degrees C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hflB1(Ts) fur mutant during aerobic growth at 42 degrees C and Feo-dependent Fe(II) transport during anaerobic growth at 42 degrees C. Temperature-resistant growth of hflB1(Ts) strains is also observed at 42 degrees C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HflB. The heat-shock response, evaluated by an htpG::lacZ fusion, is overinduced in hflB1(Ts) strains at 42 degrees C because of stabilization of sigma 32. Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hflB1(Ts) strain, and, in the hflB1(Ts) fur mutant, sigma 32 is no longer stabilized at 42 degrees C. These results reinforce the recent observation that HflB is a metalloprotease active against sigma 32 in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.

Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB.

The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host.

Leucine and serine induce mecillinam resistance in Escherichia coli.

We have previously shown that resistance to the beta-lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and RelA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of L-leucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is RelA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.

The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate.

Mecillinam, a beta-lactam antibiotic which binds specifically to penicillin-binding protein 2 (PBP2), blocks lateral cell-wall elongation, induces spherical morphology and ultimately kills bacteria. We describe here a new mecillinam-resistant mutant of Escherichia coli, the lov mutant. It possesses active PBP2, as evidenced by its rod shape in the absence of mecillinam (but not in its presence), its ability to filament when septation is inhibited, and its penicillin-binding ability. The lov mutant grows slowly but seems to regulate its macromolecular parameters properly: cell volume, RNA content (ribosome concentration), and DNA content are appropriate for the growth rate, and the growth yield is identical to that of wild type. The lov mutation is located at 41 min on the E.coli genetic map and is recessive. Certain rpsL (StrR) mutations suppress the lov mutant's mecillinam resistance. The allele specificity of the suppression suggests that the lov gene product may interact directly with the ribosomes. The lov gene product thus seems to define a link between PBP2 (the mecillinam target) and the ribosomes; we propose that this link is involved in transmitting information on the growth rate (ribosome concentration) to the peptidoglycan synthesizing apparatus.

Preliminary physiologic characterization and genetic analysis of a new Escherichia coli mutant, lov, resistant to mecillinam.

A mutant of Escherichia coli resistant to mecillinam was selected. The target of this antibiotic is penicillin-binding protein 2 (PBP2), which is active in cell wall elongation and responsible for the rod-like shape of the organism. Several observations indicate that PBP2 remains active in the mutant. For example, cells are small and rod-shaped in the absence of mecillinam but become spherical in its presence; the formation of filaments in the presence of furazlocillin shows that cell wall elongation still takes place. The mutant also has a low growth rate and small cell volume, even in rich medium. In these respects, the mutant resembles cya or crp strains (in which the cyclic adenosine monophosphate-catabolite activator protein [cAMP-CAP] complex is inactive), although its catabolic capacities are unaffected. The mutation, called lov, lies at 41 minutes on the E. coli genetic map. The phenotype is partially suppressed by the P1-mediated transduction of an rpsL ribosomal mutation conferring streptomycin resistance.

Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced.

Mecillinam, a beta-lactam antibiotic which specifically inactivates penicillin binding protein 2 (PBP2) in Escherichia coli, prevents lateral cell wall elongation, inducing spherical morphology and cell death. Two mecillinam resistant mutants, lov-1 and lovB, both able to dispense entirely with PBP2, are shown here to be affected in the aminoacyl-tRNA synthetase genes argS and alaS, respectively. Although the argS and alaS mutants grow slowly, we show that there is no correlation between mecillinam resistance and either growth rate or translation speed. A role of the ribosomes in mecillinam sensitivity, suggested by our earlier report that the lov-1 mutation is suppressed by certain rpsL(StrR) alleles affecting ribosomal protein S12, is supported by the present observation that a pseudo-streptomycin dependent mutant is mecillinam resistant in the presence of streptomycin. The argS and alaS mutants have high pools of the nucleotide ppGpp (effector of the stringent response) and the mecillinam resistance of both mutations is suppressed by a relA mutation, inactivating the ribosome-associated ppGpp synthetase and preventing ppGpp synthesis in response to aminoacyl-tRNA starvation. Furthermore, a ptacrelA' multicopy plasmid makes a wild type strain mecillinam resistant. The effect of ppGpp is probably mediated by RNA polymerase, since sublethal doses of the polymerase inhibitor rifampicin suppress mecillinam resistance in argS, alaS and ptacrelA'-bearing strains. We conclude that ppGpp regulates the transcription of a gene whose product is involved in mecillinam sensitivity, possibly as part of a chain of interacting elements which coordinate ribosomal activity with that of the PBPs.

Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ.

We recently identified RrmJ (alias FtsJ), the first encoded protein of the rrmJ-hflB heat shock operon, as an Um(2552) methyltransferase of the 23S rRNA. We now report that the rrmJ-deficient strain exhibits growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S(30) rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity. Polysome profile analysis of the rrmJ strain reveals an increase in the proportion of free 30S and 50S subunits at both 30 and 42 degrees C. These results suggest that the RrmJ-catalyzed methylation of Um(2552) in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures.

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.

The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase.

Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ.

Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH).

Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli. This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules. Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded. We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the lambdacI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI-SsrA). cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases. Loss of ClpP can be compensated for by overproducing HflB. In an in vitro system, cI108 and cI-SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus. These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases.