High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

Biosynthesis of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) and Its Regulation in Bacteria

Recent data on the biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and its regulation in bacteria are reviewed, with special emphasis on the properties and regulation of the relevant enzymes and their genes. Some conditions promoting the synthesis of PHB and PHBV by natural, mutant, and recombinant producers are considered.     

Nutritional and Metabolic Requirements for the Infection of HeLa Cells by Salmonella enterica Serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.     

Multiple Antibiotic Resistance (mar) Locus in Salmonella enterica Serovar Typhimurium DT104

In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive Salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfectants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of mar in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated. Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.     

Characterization of an Acid-Inducible Sulfatase in Salmonella enterica Serovar Typhimurium

Sulfatases of enteric bacteria can provide access to heavily sulfated mucosal glycans. In this study, we show that aslA (STM0084) of Salmonella enterica serovar Typhimurium LT2 encodes a sulfatase that requires mildly acidic pH for its expression and activity. AslA is not regulated by sulfur compounds or tyramine but requires the EnvZ-OmpR and PhoPQ regulatory systems, which play an important role in pathogenesis.     

Curcumin Increases the Pathogenicity of Salmonella enterica Serovar Typhimurium in Murine Model

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer''s patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin''s mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.     

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a “common good” for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP⁺ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP⁺ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.     

Complete Genome Sequence of the Metabolically Versatile Halophilic Archaeon Haloferax mediterranei, a Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Producer

Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.     

Epidemiology of a Salmonella enterica subsp. enterica Serovar Typhimurium Strain Associated with a Songbird Outbreak

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.     

Identification of Novel Salmonella enterica Serovar Typhimurium DT104-Specific Prophage and Nonprophage Chromosomal Sequences among Serovar Typhimurium Isolates by Genomic Subtractive Hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments—irsA, the HldD homologue, and three fragments with sequence similarity to prophages—were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.     

Effects of Low Dissolved-Oxygen Concentrations on Poly-(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Production by Alcaligenes eutrophus

The bacterial copolyester poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced with Alcaligenes eutrophus DSM 545 from glucose and sodium propionate in a fed-batch fermentation with both nitrogen limitation and low dissolved-oxygen concentrations. When the dissolved-oxygen content was kept between 1 and 4% of air saturation during the polymer accumulation phase, the yield of 3-hydroxybutyrate (3HB) monomer from glucose was not affected, but the propionate-to-3-hydroxyvalerate (3HV) monomer yield was two to three times (0.48 to 0.73 mol of 3HV mol of propionate consumed(sup-1)) that observed in a control experiment (0.25 mol mol(sup-1)), where the accumulation-phase dissolved-oxygen concentration was 50 to 70% of air saturation. The overall polymer productivity of the fermentation was somewhat decreased by low dissolved-oxygen contents, owing to a slower 3HB production rate. The effect of a low dissolved-oxygen concentration is probably attributable to a reduction of the oxygen-requiring decarbonylation of propionyl-coenzyme A (CoA) to acetyl-CoA.     

Short-Term Signatures of Evolutionary Change in the Salmonella enterica Serovar Typhimurium 14028 Genome

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis in humans and a typhoid-like disease in mice and is often used as a model for the disease promoted by the human-adapted S. enterica serovar Typhi. Despite its health importance, the only S. Typhimurium strain for which the complete genomic sequence has been determined is the avirulent LT2 strain, which is extensively used in genetic and physiologic studies. Here, we report the complete genomic sequence of the S. Typhimurium strain 14028s, as well as those of its progenitor and two additional derivatives. Comparison of these S. Typhimurium genomes revealed differences in the patterns of sequence evolution and the complete inventory of genetic alterations incurred in virulent and avirulent strains, as well as the sequence changes accumulated during laboratory passage of pathogenic organisms.The genomes of related bacteria can differ in three ways: (i) gene content, where one bacterial species or strain harbors genes absent from the other organism; (ii) nucleotide substitutions within largely conserved DNA sequences, which can result in amino acid changes in orthologous proteins, form pseudogenes, and promote distinct expression patterns of genes present in the two organisms; and (iii) changes in gene arrangement, caused by inversions and translocations. These differences have been observed not only across bacterial species but also among strains belonging to the same species. Recent genomic analyses have revealed that many bacterial pathogens of humans are virtually monomorphic (1) and exhibit very limited sequence diversity, raising questions about the nature of the genetic changes governing distinct behaviors. Furthermore, several bacterial pathogens that have been subjected to extensive passage in the laboratory display altered virulence characteristics, but the genetic basis for these alterations remains largely unknown. Here, we address both of these questions by determining and analyzing the genome sequences of closely related isolates of Salmonella enterica serovar Typhimurium, a Gram-negative pathogen that has been used as a preeminent model to investigate basic genetic mechanisms (2, 8, 46, 59), as well as the interaction between bacterial pathogens and mammalian hosts (11, 41).The genus Salmonella is divided into two species: Salmonella bongori and Salmonella enterica, which together comprise over 2,300 serovars differing in host specificity and the disease conditions they promote in various hosts. For example, S. enterica serovar Typhi is human restricted and causes typhoid fever, whereas serovar Typhimurium is a broad-host-range organism that causes gastroenteritis in humans and a typhoid-like disease in mice. Although the complete genome sequences of 15 Salmonella enterica strains are available, there is only a single representative of S. Typhimurium—strain LT2 (31). Despite its wide application in genetic analysis, strain LT2 is highly attenuated for virulence in both in vitro and in vivo assays (52, 56), leading many investigators to use other S. Typhimurium isolates to examine the genetic basis for bacterial pathogenesis (11, 14, 16).Over 300 virulence genes (3, 5, 47) have already been identified in Salmonella enterica serovar Typhimurium 14028 (now termed S. enterica subsp. enterica serovar Typhimurium ATCC 14028), which is a descendant of CDC 60-6516, a strain isolated in 1960 from pools of hearts and livers of 4-week-old chickens (P. Fields, personal communication). Whereas strain 14028 has been typed as LT2, a designation based on phage sensitivity (27), the two strains were isolated from distinct sources decades apart, which makes their genealogy and exact relationship obscure. A derivative of the original 14028 strain with a rough colony morphology (due to changes in O-antigen expression) was designated 14028r to distinguish it from the original smooth strain, renamed 14028s, and was used in a genetic screen for Salmonella virulence genes because it retained lethality for mice and the ability to survive within murine macrophages. Strain 14028 was also used for the identification of Salmonella genes that were specifically expressed during infection of a mammalian host (30). Both 14028 and LT2 possess a 90-kb virulence plasmid promoting intracellular replication and systemic disease (14), but they differ in their prophage contents, as is often the case among S. Typhimurium strains (12, 13).To identify the individual changes that differentiate S. Typhimurium strains and to assess the nature of variation that arises during laboratory storage and passage, we determined the genome sequence of strain 14028s. This genome was then used as a reference for sequencing its progenitors, including the original source strain CDC 60-6516 and the earliest smooth and rough variants. Our analysis uncovered the genomic differences that arose during the past decades of laboratory cultivation and showed that derivatives with different virulence potentials can follow distinct patterns of sequence evolution.     

Selection of Orphan Rhs Toxin Expression in Evolved Salmonella enterica Serovar Typhimurium

Clonally derived bacterial populations exhibit significant genotypic and phenotypic diversity that contribute to fitness in rapidly changing environments. Here, we show that serial passage of Salmonella enterica serovar Typhimurium LT2 (StLT2) in broth, or within a mouse host, results in selection of an evolved population that inhibits the growth of ancestral cells by direct contact. Cells within each evolved population gain the ability to express and deploy a cryptic “orphan” toxin encoded within the rearrangement hotspot (rhs) locus. The Rhs orphan toxin is encoded by a gene fragment located downstream of the “main” rhs gene in the ancestral strain StLT2. The Rhs orphan coding sequence is linked to an immunity gene, which encodes an immunity protein that specifically blocks Rhs orphan toxin activity. Expression of the Rhs orphan immunity protein protects ancestral cells from the evolved lineages, indicating that orphan toxin activity is responsible for the observed growth inhibition. Because the Rhs orphan toxin is encoded by a fragmented reading frame, it lacks translation initiation and protein export signals. We provide evidence that evolved cells undergo recombination between the main rhs gene and the rhs orphan toxin gene fragment, yielding a fusion that enables expression and delivery of the orphan toxin. In this manner, rhs locus rearrangement provides a selective advantage to a subpopulation of cells. These observations suggest that rhs genes play important roles in intra-species competition and bacterial evolution.