The Rcs Stress Response and Accessory Envelope Proteins Are Required for De Novo Generation of Cell Shape in Escherichia coli

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Actinomycin Resistance and Actinomycin Excretion in a Mutant of Escherichia coli

A mutant of Escherichia coli resembles its parent in taking up actinomycin after treatment with ethylenediaminetetraacetic acid but differs in that it survives this uptake and excretes actinomycin at an increased rate.     

Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.     

Survival of Low-pH Stress by Escherichia coli O157:H7: Correlation between Alterations in the Cell Envelope and Increased Acid Tolerance

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 × 10⁴ CFU ml⁻¹ were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.     

Identification of microRNA-Size, Small RNAs in Escherichia coli

Noncoding small regulatory RNA molecules control gene expression and microRNAs provide one of the best examples in eukaryotes. However, bacterial RNAs of comparable size to eukaryotic microRNAs have received little attention. Here, we demonstrate the existence of microRNA-size, small RNAs (msRNAs) in the model bacterium Escherichia coli. We examined the small RNAs in E. coli using a deep sequencing approach, and analyzed 33.2 million small RNA clone reads after size fractionation. Bioinformatic analysis of the whole set revealed more than 400 individual msRNA species. The cellular contents of selected highly expressed msRNAs were verified by quantitative RT-PCR and northern blotting. Although, the functional significance of these RNAs is unclear, their high abundance suggests that they may play specialized roles in bacteria, analogous to miRNAs in eukaryotes.     

Control of Acid Resistance in Escherichia coli

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.     

植物小RNAs及其抗胁迫机制研究进展

植物非编码小RNA(sRNAs)主要分为三类:微小RNA(m iRNAs)、小干扰RNA(siRNAs)和长小片段干扰RNA(lsiRNAs)。三者的生物合成和作用机制有所不同,但他们主要都通过介导靶mRNAs的剪切或抑制其翻译来调控基因的表达。这篇文章主要介绍小RNA研究的最新进展,并重点阐述其在非生物和生物胁迫中发挥的作用,如应对矿质元素缺乏、氧化胁迫、ABA胁迫以及病原菌入侵等生理过程。     

Enhanced Resistance to Nitrosoguanidine Killing and Mutagenesis in a DNA Gyrase Mutant of Escherichia coli

The role of DNA gyrase in handling DNA damages induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined with two Escherichia coli strains, KL161 and KL166. The two strains are isogenic except that KL166 harbors a mutation at the nalA (gyrA) locus which specifies one of the two subunits of DNA gyrase. We treated the two strains with several different types of mutagenic agents and found the nalA strain to be highly resistant to MNNG-induced killing and mutagenic effects as compared with the parental strain. The MNNG resistance was specific, since the two strains were about equally sensitive to methyl methane sulfonate, ethyl methane sulfonate, and UV and gamma radiations. We pulse-labeled the two strains with [(3)H]uridine and (14)C-amino acids after MNNG treatment to analyze RNA and protein synthetic rates. The pulse-labeled proteins were also separated on polyacrylamide gels. The results show that pulse-labeled RNA and proteins persisted in the nalA strain but declined rapidly in the parental strain after MNNG treatment. We compared membrane-free nucleoid preparations from the two strains by sucrose density gradient centrifugation and found a difference in nucleoid organization between the two strains. The nucleoid of the nalA strain, unlike that of the parental strain, may have a highly ordered structure, as indicated by its resistance to ethidium bromide-induced relaxation. The ability of the two strains to express an adaptive response to MNNG was determined. We found that the resistance to MNNG killing and mutagenesis by the nalA strain cannot be further increased by adaptive treatment. These results suggest that an alteration in DNA gyrase may have profound effects on E. coli chromosome organization and base methylation by MNNG.     

Iron-binding Proteins in Milk and Resistance to Escherichia coli Infection in Infants

Human milk contains large quantities of iron-binding protein, of which the greater proportion is lactoferrin, though small amounts of transferrin are also present. Three samples of human milk with unsaturated iron-binding capacities of between 56 and 89% had a powerful bacteriostatic effect on Escherichia coli O111/B4. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. Purified human lactoferrin, in combination with specific E. coli antibody, strongly inhibited the growth of E. coli, and this effect was also abolished by saturating the lactoferrin with iron.Guinea-pig milk also contains lactoferrin and transferrin. Newly born guinea-pigs fed on an artificial diet and dosed with E. coli O111 had higher counts of E. coli O111 in the intestine than suckled animals. The apparent suppressive effect of guinea-pig milk on E. coli in the intestine could be reversed by feeding the iron compound haematin. It seems that iron-binding proteins in milk may play an important part in resistance to infantile enteritis caused by E. coli.     

Properties of a Deoxyribonucleic Acid Ligase Mutant of Escherichia coli: X-Ray Sensitivity

A deoxyribonucleic acid ligase-deficient mutant is X-ray sensitive relative to the parent strain, suggesting that deoxyribonucleic acid ligase functions in repair of X-ray-induced, single-strand scissions.     

Regulation of Cell Division in a Temperature-Sensitive Division Mutant of Escherichia coli

Escherichia coli fil ts forms multinucleate filaments when suspensions of about 10(7) organisms per ml are shifted from 37 to 43 C in rich medium. Occasional septation continues, chiefly at the poles, and immediately becomes more frequent when the filaments are returned to 37 C. The addition of chloramphenicol (200 mug/ml) at either temperature initially stimulates the formation of polar septa. When very dilute suspensions of the strain (<10(6) organisms per ml) are shifted to the restrictive temperature, the inhibition of septation is more complete and only seldom reversible. Conversely, cell division is little affected when suspensions of >10(8) organisms per ml, or microcolonies of several hundred organisms on agar, are incubated at 43 C; evidence is presented that this is a consequence of a slight reduction in the mutant's growth rate. In certain media, septation is blocked irreversibly by even brief exposure to 43 C, after which cell elongation without division proceeds at 37 C for some hours. Several findings, when considered together, suggest that the cytoplasmic membrane is normal at the restrictive temperature, and that the block in septation is caused by a defect in the cell wall: it is largely overcome by NaCl, but not by sucrose; in some circumstances the filaments become swollen and develop localized bulges in the wall, yet the membrane remains intact and retains its selective permeability; lastly, the strain is insensitive to deoxycholate at both temperatures. The mutation has been mapped between arg B and thr, at a locus which appears to be distinct from others known primarily to influence cell division.     

Ribosomal Proteins Involved in the Suppression of Streptomycin Dependence in Escherichia coli

Suppression of streptomycin dependence in Escherichia coli strain K-114, a spectinomycin-sensitive strain, is correlated with modification of 30S ribosomal protein P4, the component modified in spectinomycin-resistant mutants. The mutant is unusual in that reversion from dependence has previously been correlated only with modification in 30S protein P4a. Introduction into K-114 of another mutation conferring spectinomycin resistance results in a further alteration in protein P4.     

Transfer RNAs Mediate the Rapid Adaptation of Escherichia coli to Oxidative Stress

Translational systems can respond promptly to sudden environmental changes to provide rapid adaptations to environmental stress. Unlike the well-studied translational responses to oxidative stress in eukaryotic systems, little is known regarding how prokaryotes respond rapidly to oxidative stress in terms of translation. In this study, we measured protein synthesis from the entire Escherichia coli proteome and found that protein synthesis was severely slowed down under oxidative stress. With unchanged translation initiation, this slowdown was caused by decreased translation elongation speed. We further confirmed by tRNA sequencing and qRT-PCR that this deceleration was caused by a global, enzymatic downregulation of almost all tRNA species shortly after exposure to oxidative agents. Elevation in tRNA levels accelerated translation and protected E. coli against oxidative stress caused by hydrogen peroxide and the antibiotic ciprofloxacin. Our results showed that the global regulation of tRNAs mediates the rapid adjustment of the E. coli translation system for prompt adaptation to oxidative stress.     

Roles of the Escherichia coli Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42°C when combined with the dnaK756 or groES30 alleles, while the Δibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.     

Ultraviolet- and X-Ray-Induced Responses of a Deoxyribonucleic Acid Polymerase-Deficient Mutant of Escherichia coli

Escherichia coli K-12, polAl(-) is a mutant strain whose extracts are deficient in Kornberg deoxyribonucleic acid (DNA) polymerase activity. We have compared the mutant and parental strains on the basis of a number of responses to ultraviolet (UV) and X-irradiation. For both types of radiation, the mutant is more sensitive by approximately the same factor as measured by reduction in colony formation, depression of DNA synthesis, and enhancement of DNA degradation. The rate of repair of X-ray-induced single-strand breaks in the mutant is also slower, as is the repair of breaks after excision repair of UV damage. On the other hand, the mutant has a significant capability to reactivate UV-irradiated lambda phage, although it is almost totally deficient in the ability to carry out UV reactivation. The data indicate that the polAl mutation leaves the cells with some ability to perform excision and strand-rejoining repair but that an exonuclease, whose identity remains obscure, is the agent responsible for the extensive breakdown of the DNA in polAl(-) cells after irradiation.     

Small Heat Shock Proteins Protect Electron Transport in Chloroplasts and Mitochondria During Stress

Evidence indicates that small heat-shock proteins (Hsps) areinvolved in stress tolerance, but the specific cell componentsor functions that small Hsps protect or repair are mostly unidentified.We recently showed that the chloroplast small Hsps of higherplants (1) are produced in response to many environmental stresses(e.g., heat, oxidative, and high-light stress); and (2) protect(but do not repair) photosynthetic electron transport in vitroduring stress, specifically by interacting with the oxygen-evolving-complexproteins of Photosystem II (PSII) within the thylakoid lumen.However, in vivo evidence of the importance of these Hsps tophotosynthetic stress tolerance is lacking. Here we report positiverelationships between chloroplast small Hsp production and PSIIthermotolerance in (1) a heattolerant genotype of Agrostis palustris(bentgrass) and a heat sensitive genotype which lacks one ormore chloroplast small Hsps produced by the tolerant genotype;(2) ecotypes of Chenopodium album (lambs quarters) from thenorthern vs. southern U.S. (New York vs. Georgia); and (3) nineLycopersicon (tomato) cultivars/species differing in heat tolerance.These in vivo results are consistent with our previous in vitroobservations and indicate that genetic variation in productionof the chloroplast small Hsp is an important determinant ofphotosynthetic and, thereby, whole-plant thermotolerance. Recently,we showed that the mitochondrial small Hsp of plants protectsrespiratory (specifically Complex I) electron transport in vitroduring heat stress, and here we present evidence for previouslyunidentified small Hsps in mitochondria of mammal (rat) cellswhich also protect Complex I during heat stress. These resultssuggest that the mitochondrial small Hsps, like the small chloroplastHsps, are general stress proteins that contribute significantlyto cell and organismal stress tolerance.