Methylotrophic growth of a mutant strain of the acetogenic bacterium Eubacterium limosum that uses acetate as co-substrate in the absence of CO2

Abstract Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col⁻, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col⁻ and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae and enhanced E. coli F-18Col⁻ colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX , which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two proteins (22 kDa and 26 kDa) encoded by genes immediately adjacent to leuX .     

Altered colonizing ability for mouse large intestine of a surface mutant of a human faecal isolate of Escherichia coli

Escherichia coli F-17 Sr a human faecal isolate, is resistant to the T-series of bacteriophages (i.e. T2 to T7). A T2-sensitive mutant of E. coli F-17 Sr was isolated following acriflavin treatment. This mutant, E. coli F-17 Sr Ts was found to be sensitive to the entire T-series of phages. E. coli F-17 Sr and E. coli F-17 Sr Ts did not differ quantitatively in total LPS content. However, analysis of LPS revealed that a large fraction of E. coli F-17 Sr Ts was devoid of O-side-chains. This accounted for the sensitivity of this strain to bacteriophages T3, T4, and T7. In addition, E. coli F-17 Sr Ts contained only about half the amount of capsular material contained by E. coli F-17 Sr accounting for the sensitivity of the mutant to bacteriophages T2, T5, and T6. Although the two strains colonized equally well when fed individually to streptomycin-treated mice, when fed simultaneously to streptomycin-treated mice, E. coli F-17 Sr Ts colonized at a level of about 1 x 10(8) cells (g faeces)-1, whereas E. coli F-17 Sr colonized at only 1 x 10(4) cells (g faeces)-1. These studies suggest that bacterial cell surface components modulate the large intestine colonizing ability of E. coli F-17 Sr in the mouse large intestine.     

Escherichia coli K-12 ferrous iron uptake mutants are impaired in their ability to colonize the mouse intestine

Abstract The streptomycin-treated mouse colonization model was used to investigate the role of the Fe²⁺ uptake system (Feo) of Escherichia coli K12 in the colonization of the mouse intestine. Mutants impaired in the uptake of Fe²⁺ ions were shown to be deficient also in their colonization ability. Both enterochelin-producing and enterochelin-nonproducing Escherichia coli feo mutants were unable to colonize the mouse intestine. These results demonstrated that Fe(II) is an essential source of iron for E. coli grown in the intestine.     

Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ . In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coli growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

Comparison of the Activities of Extracts of Escherichia coli and Salmonella typhimurium in Amino Acid Incorporation

We have compared the amino acid incorporating activities of extracts of Escherichia coli and Salmonella typhimurium in in vitro protein-synthesizing systems directed by bacterial messenger ribonucleic acid (mRNA) of both species and by the genomes of coliphages Qbeta and f2. E. coli and S. typhimurium extracts translate both homologous and heterologous bacterial mRNAs at comparable rates. S. typhimurium extracts translate phage RNAs only 10 to 15% as fast as E. coli extracts do. The presence of glucose in the growth medium increases the activity of S. typhimurium extracts three- to fourfold in the phage RNA-directed systems. Glucose has a much more limited effect on the activities of E. coli extracts. We show that similar amounts of phage RNA-ribosome complexes are formed in both the E. coli and the S. typhimurium systems, indicating that the different activities observed may be attributed to different rates of peptide elongation or to the formation of complexes at different sites on the RNA strand.     

A strain of Enterococcus faecium (18C23) inhibits adhesion of enterotoxigenic Escherichia coli K88 to porcine small intestine mucus

Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if Enterococcus faecium 18C23 has the ability to inhibit the adhesion of Escherichia coli K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% of E. faecium 18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living E. faecium 18C23 culture efficiently inhibited the adhesion of E. coli K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of E. coli K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 10(9) CFU or more of living E. faecium 18C23 culture per ml was added simultaneously with E. coli to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of E. coli K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of E. coli K88ac by E. faecium 18C23 or its supernatant might occur through steric hindrance.     

In vitro antibacterial activity of Lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria

AIMS: The purpose of this study was to investigate in vitro the antibacterial activity of the Lactobacillus helveticus strain KS300 against vaginosis-associated bacteria including Gardnerella vaginalis and Prevotella bivia, uropathogenic Escherichia coli, and diarrhoeagenic Salmonella enterica serovar Typhimurium. METHODS AND RESULTS: The KS300 strain inhibited the growth of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. After direct co-culture, data show that the Lactobacillus strain decreased the viability of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. The adhering KS300 strain inhibited the adhesion of G. vaginalis DSM 4944 and uropathogenic Dr-positive E. coli IH11128 onto HeLa cells. Moreover, the KS300 strain inhibited the internalization of uropathogenic Dr-positive E. coli IH11128 within HeLa cells and S. typhimurium SL1344 within Caco-2/TC7 cells. CONCLUSIONS: The findings demonstrate that L. helveticus strain KS300 is adhesive onto cultured human cells and has antagonistic activities against vaginosis-associated, uropathogenic and diarrhoeagenic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhering L. helveticus strain KS300 is a potential probiotic strain displaying a strain-specific array of in vitro antibacterial activities.     

Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.     

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion.