Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.Entomopathogenic Heterorhabditis and Steinernema nematodes are safe and effective bioinsecticides for the biological control of many economically important pests (9). The infective juveniles (IJs) of these nematodes harbor Photorhabdus or Xenorhabdus bacteria as symbionts in their intestines. The IJ nematodes properly maintain and carry the bacteria needed for killing insects and providing a suitable environment for the reproduction of new vectors (5, 8). Different bacterial isolates differ in their ability to support in vitro monoxenic cultures of nonhost nematodes (2, 7, 13) and to retain the bacterial cells in the IJ intestines (2, 8, 11).Strains of Photorhabdus and Xenorhabdus spp. not only show insecticidal activities toward different insects (3, 4, 21) but also exhibit nematicidal activities against nematodes (14, 16, 17). The trans-specific nematicidal activity of Photorhabdus luminescens subsp. akhurstii LN2, a normal symbiont of Heterorhabditis indica LN2 against Heterorhabditis bacteriophora H06, was previously observed (12). The LN2 bacteria may secrete unidentified toxic factors that are lethal to the H06 nematodes. However, the genes of these bacteria involved in the trans-specific nematicidal activities have not been reported.This paper describes the identification, through Tn5 mutagenesis and characterization, of a novel P. luminescens LN2 gene involved in nematicidal activity against the H06 IJs. The colonization of the green fluorescent protein (GFP)-labeled mutant cells in H06 IJ intestines was examined.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.     

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Lactobacillus Strain Ecology and Persistence within Broiler Chickens Fed Different Diets: Identification of Persistent Strains

Lactobacilli are autochthonous residents in the chicken gastrointestinal tract, where they may potentially be used as probiotics, competitive exclusion agents, or delivery vehicles. The aim of this study was to use an in vivo model to investigate the effect of diet and competing lactic acid bacteria on the colonization of inoculated Lactobacillus strains, with the goal of identifying strains which can consistently colonize or persist for an extended period of several weeks. Chicken-derived Lactobacillus strains were genetically marked with rifampin resistance and administered on day 0 to chickens fed either a normal commercial diet or a specially formulated high-protein diet. Chickens fed the high-protein diet were also coinoculated with two different mixes of additional lactic acid bacteria. Enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) was used to identify rifampin-resistant isolates recovered from chickens. Three strains, belonging to the species Lactobacillus agilis, Lactobacillus crispatus, and Lactobacillus vaginalis, were commonly reisolated from the chickens on both diets at days 21 and 42. The ability of these strains to persist was confirmed in a second chicken trial. All three strains persisted throughout the production period in the chickens fed a commercial diet, while only the L. agilis and L. vaginalis strains persisted in the chickens fed the high-protein diet. In both in vivo trials, competing lactic acid bacteria modified representation of the strains recovered, with all three stains capable of competing in the presence of one or both mixes of coinoculated strains. The in vivo model successfully identified three persistent strains that will be characterized further.The ecology of the chicken gastrointestinal tract (GIT) has been studied in depth using both culture-dependent (5, 7, 21, 40) and -independent methods (2, 3, 7, 33, 54). These studies have revealed that lactobacilli are autochthonous residents in chickens, where they predominate in the proximal GIT and are present but less abundant within the distal GIT (52). The most commonly identified Lactobacillus species are Lactobacillus crispatus, Lactobacillus reuteri, and Lactobacillus salivarius (1, 7, 15, 26, 28). A detailed understanding of the relationship between these bacteria and their host under different dietary and environmental conditions will facilitate the development of lactobacilli for various applications directed toward increasing broiler production efficiency and improving chicken health.Since the withdrawal of antimicrobial growth promoters from chicken feed in Europe, the incidence of necrotic enteritis (NE) has increased (9, 51). Consequently, there is a need to develop alternative methods for controlling the causative agent of NE, Clostridium perfringens, in the chicken GIT. Lactobacilli are excellent candidates for alternative control methods due to their autochthonous nature and dominance of the upper GIT microbiota, particularly within the small intestine where NE occurs. Their potential utility in the control of NE has been demonstrated, with several strains of Lactobacillus showing some efficacy as probiotics to decrease C. perfringens carriage within the small intestine of chickens (14, 25, 31, 37, 47). Lactobacilli are also excellent candidates as mucosal delivery vectors designed to express bioactive peptides in situ to reduce colonization by C. perfringens. The use of lactobacilli and other lactic acid bacteria (LAB) as live delivery vectors for therapeutic proteins has recently been reviewed (6, 53), but few studies have been conducted in chickens (46, 55, 56). An attenuated Salmonella enterica serovar Typhimurium delivery vector targeting C. perfringens has provided partial protection against experimental NE challenge (35, 57). Identification of Lactobacillus strains for use as delivery vectors, competitive exclusion agents, or probiotics is complicated by the difficulty in selecting truly autochthonous strains capable of reliably and consistently colonizing the chicken GIT upon subsequent inoculation.Traditionally, strain selection for in vivo applications has involved several in vitro characterization assays, including assays of aggregation, coaggregation, cell wall hydrophobicity, acid tolerance, bile salt tolerance, adhesion to epithelial cell lines, and antimicrobial activity (23, 32, 34, 44, 49). While these assays can be used to reduce the number of strains examined, they may also bias the selection of strains and could potentially overlook strains which may be competitive or have other desirable characteristics in vivo. One of the limitations of in vivo screening of lactobacilli is the need for reliable high-throughput screening techniques to identify and track persistent strains. Recently, our group reported the application of enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) to simultaneously type large numbers of Lactobacillus isolates from the chicken GIT to the species and strain level (48).The primary aim of this study was to use a new, direct, in vivo screening method to examine the ecology of inoculated Lactobacillus strains in chickens fed different diets (high protein versus commercial). A second aim of this study was to determine the ecological effect of coinoculating two different mixes of competing LAB in chickens fed a high-protein diet, which predisposes chickens to develop NE. Inoculated strains were marked with rifampin resistance (Rif^r), and ERIC-PCR was used to identify strains isolated from the chickens at days 21 and 42. Three persistent strains were identified in the initial trial and selected for further characterization in a subsequent experiment, in which two strains persisted in chickens fed the high-protein diet and all three persisted in chickens fed the commercial diet. In general, competing LAB modified strain representation and, in some cases, facilitated colonization of some strains. These three persistent strains will be further characterized as potential vectors to be used in the antibiotic-free control of NE.     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject''s stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.Helicobacter pylori, a gram-negative bacterium, is remarkable for its ability to persist in the human stomach for decades. Colonization with H. pylori increases risk for peptic ulcer disease and gastric adenocarcinoma (53, 70) and elicits a vigorous immune response (15). The persistence of H. pylori occurs in a niche in the human body previously considered inhospitable to microbial colonization: the acidic stomach replete with proteolytic enzymes.H. pylori strains exhibit substantial genetic diversity, including extensive variation in the presence, arrangement, order, and identity of genes (2, 4-7, 25, 51, 74). Furthermore, analyses of multiple single-colony H. pylori isolates from separate stomach biopsy specimens of individual patients have demonstrated diversity, both within hosts (27, 65), and over time (36). The mechanisms that generate H. pylori genetic diversity may be among the factors that enable persistence in this environment (3, 28).While the natural ability of H. pylori for transformation and recombination may explain some of the intra- and interhost genetic variation observed in this bacterium (43), point mutations and interspecies recombination alone are not sufficient for explaining the extent of the variation in H. pylori (14, 32). The initial genomic sequencing of H. pylori strains 26695 and J99 (6, 72) revealed large amounts of repetitive DNA (1, 59). DNA repeats in bacteria are associated with mechanisms of plasticity, such as phase variation (49, 67); slipped-strand mispairing (41, 46); and increased rates of recombination, deletion, and insertion (17, 60, 62). Because many of the recombination repair and mismatch repair mechanisms common in bacteria are absent or modified in H. pylori (28-30, 56, 76), this organism may be particularly susceptible to the diversifying effects of repetitive DNA. In fact, loci in the H. pylori genome containing repetitive DNA have been shown to exhibit extensive inter- and intrahost variation (9, 10, 28, 37).We hypothesized that identification of repetitive DNA hotspots in H. pylori would allow the recognition of genes whose variation could aid in persistence. To examine this hypothesis, we conducted in silico analyses to identify open reading frames (ORFs) enriched for DNA repeats and then used a combination of sequence analyses and immunoassays to examine the patterns associated with the specific repetitive DNA observed. Our approach led to the realization that a previously identified H. pylori-specific gene family (19, 52) exhibits extensive genetic variation at multiple levels.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Horizontal Chromosome Transfer,a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.Fungi produce a huge variety of secondary metabolites. Some plant-pathogenic fungi, especially necrotrophic pathogens that kill plant cells during invasion, produce phytotoxic metabolites to impair host tissue functions (20, 30, 42, 47). Phytotoxins produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites that exert toxic effects on host plants. Among these phytotoxins, host-specific toxins (HSTs) are critical determinants of pathogenicity or virulence in several plant-pathogen interactions (13, 30, 33, 40, 42, 47, 49).Recent advances in molecular biological techniques for fungi have led to the identification of fungal genes involved in pathogenesis, as exemplified by those used in the biosynthesis of toxic secondary metabolites, such as HSTs. Genes involved in the biosynthesis of secondary metabolites are typically clustered in filamentous fungi, including plant pathogens (20, 24, 44). The origins and evolutionary processes of these gene clusters, however, are largely unknown. Analysis of the arrangement and sequences of genes in the clusters would shed light on how the clusters themselves and their ability to produce toxic secondary metabolites evolved (20, 24, 44).The involvement of horizontal gene transfer (HGT) in the evolution of fungal secondary-metabolite gene clusters has been discussed (34, 44). HGT events are well known in prokaryotes (21, 29), and the genomic regions that have undergone HGT are referred to as pathogenicity or genomic islands (7). In prokaryotes, the mechanisms of HGT are also associated with conjugation, transformation, and transduction (21, 29). Although these transfer mechanisms are generally unknown in eukaryotes such as fungi, interspecific transfer of a virulence gene encoding the production of a critical toxin has been reported in Pyrenophora tritici-repentis (14). There is also clear evidence of recent lateral gene transfer of the ToxA gene from Stagonospora nodorum to P. tritici-repentis (14, 30).In Alternaria alternata plant pathogens (37), we have shown that all strains of the A. alternata pathotypes harbor small extra chromosomes of less than 1.7 Mb, whereas nonpathogenic isolates do not have these small chromosomes (5). A cyclic peptide synthetase gene, AMT, which is involved in host-specific AM toxin biosynthesis of the apple pathotype of A. alternata, was located on a small chromosome of 1.1 to 1.7 Mb, depending on the strain (22, 23). The AF toxin biosynthesis gene cluster was also present on a single small chromosome of 1.05 Mb in the strawberry pathotype of A. alternata (18). Based on biological and pathological observations, those small chromosomes were regarded as supernumerary chromosomes, or conditionally dispensable chromosomes (CDCs) (10, 18, 22). Fungal supernumerary chromosomes, which are not important for normal growth but confer advantages for colonizing an ecological niche, such as infecting host plants, are regarded as CDCs (21). The functions and pathological roles of CDCs have been studied in the pea pathogen Nectria haematococca (11, 17, 25, 32, 43, 46).The origin and evolution of CDCs have been intriguing issues in the study of plant-microbe interactions. The supernumerary chromosomes of certain strains of N. haematococca have been suggested to have a different evolutionary history than essential chromosomes (ECs) in the same genome, and they might have been introduced into the genome by horizontal transfer from another strain (10, 12, 36). In Colletotrichum gloeosporioides, the 2-Mb supernumerary chromosome was transferred from a biotype A strain to a vegetative incompatible biotype B strain (19, 31). Transfer of the chromosome, however, did not affect the pathogenicity of the recipient fungus, perhaps because it did not harbor pathogenicity genes (19, 31). These results suggest that supernumerary chromosomes of fungi might have the capacity for horizontal transfer across an incompatibility barrier between two distinct strains.AAL toxins are HSTs produced by the tomato pathotype of A. alternata (synonym A. alternata f. sp. lycopersici, synonym Alternaria arborescens), the causal agent of Alternaria stem canker disease in tomatoes, which causes severe necrosis of susceptible tomato cultivars (15, 26, 35). AAL toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and termed sphinganine-analogue mycotoxins. AAL toxins and fumonisins are sphinganine-analogue mycotoxins, which are toxic to some plant species and mammalian cells (16, 48). They cause apoptosis in susceptible tomato cells and mammalian cells by inhibiting ceramide biosynthesis (9, 41, 45). In the tomato pathotype of A. alternata-tomato interactions, a major factor in pathogenicity is the production of host-specific AAL toxins capable of inducing cell death only in susceptible cultivars (3, 9, 48).In this study, we describe evidence showing that the ability to produce the host-specific AAL toxin and to infect host tomato plants could potentially be distributed among a population of strains of the A. alternata tomato pathotype by horizontal transfer of an entire pathogenicity chromosome of the pathogen.     

Comparison and Utilization of Repetitive-Element PCR Techniques for Typing Lactobacillus Isolates from the Chicken Gastrointestinal Tract

Three repetitive-element PCR techniques were evaluated for the ability to type strains of Lactobacillus species commonly identified in the chicken gastrointestinal tract. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) produced species- and strain-specific profiles for Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri isolates. The technique typed strains within these species equally as well as pulsed-field gel electrophoresis. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Subsequently, ERIC-PCR was used to type Lactobacillus species diversity of a large collection of isolates derived from chickens grown under commercial and necrotic enteritis disease induction conditions. This study has illustrated, for the first time, that there is great strain diversity within each Lactobacillus species present and has revealed that chickens raised under commercial conditions harbor greater species and strain diversity than chickens raised under necrotic enteritis disease induction conditions.Lactobacilli are normal inhabitants within the microflora of the chicken gastrointestinal tract (GIT) (27, 39). Species frequently identified within the chicken GIT include Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri (1, 7, 27). The first three of these species are members of the Lactobacillus acidophilus complex (LAC) (22, 32), a closely related group of species which are difficult to differentiate using traditional techniques, such as physiological and biochemical tests (34). While molecular methods, such as DNA-DNA hybridization (22), ribotyping (71), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19), randomly amplified polymorphic DNA PCR (19), and 16S rRNA gene sequencing (41), have been used with some success, many of these techniques are not readily adaptable to high-throughput applications required for large-scale ecological studies.While numerous studies have reported Lactobacillus species distribution within the chicken GIT, the strain diversity within species has not been explored. Previously, Hagen et al. (28) investigated L. gallinarum isolates present within the crops of commercial chickens, revealing a high level of strain diversity among the isolates examined (17 strains represented among 38 isolates). These results indicate that there could be great diversity within and among the lactobacilli present within the chicken GIT. Examining and typing large numbers of lactobacilli from the chicken GIT to the strain level may facilitate a better understanding of microflora dynamics and niche competition. These studies may also result in the identification of strains which could be used in competitive exclusion applications and potentially in the development of probiotics or live vectors for the delivery of therapeutic recombinant proteins to specific sites within the chicken GIT.Lactobacilli have been proposed as possible competitive exclusion agents or probiotics (30, 36, 42) against Clostridium perfringens, the causative agent of necrotic enteritis (NE) in broiler chickens. The withdrawal of antimicrobial growth promoters in Europe has led to an increase in the incidence of NE (10, 64), prompting the investigation of alternative methods for controlling C. perfringens in the broiler chicken GIT. Various models have been developed to study NE under research conditions, as recently reviewed by Dahiya et al. (15). The conditions used in these NE models and the effects they have on GIT microflora may adversely impact the identification of antimicrobial alternatives, such as probiotic strains, for use within commercial broiler chickens. Application of a high-throughput typing method capable of distinguishing species and strains is needed to determine if differences exist between the Lactobacillus populations of chickens raised under NE and commercial conditions.While pulsed-field gel electrophoresis (PFGE) has been used widely for genotyping Lactobacillus strains (49, 50, 65), it is time-consuming, labor-intensive, expensive, and suitable only for low-throughput analysis of isolates (54, 65). In contrast, repetitive-element PCR (Rep-PCR) has been developed for genotypically fingerprinting bacteria and is a fast and reliable high-throughput genotyping system (66, 67). Rep-PCR has been used successfully to identify strains of a variety of genera (33, 44, 48, 55). Several Rep-PCR primers, including the repetitive extragenic palindromic (REP) primers (6, 9, 16, 65), the enterobacterial repetitive intergenic consensus (ERIC) primers (6, 65), and the (GTG)₅ primer (24, 40, 62), have been used in the typing of lactobacilli in studies which have generally focused on species important to the dairy industry and food fermentations. Very few studies have examined the application of Rep-PCR to species commonly identified in the chicken GIT (62, 65). To our knowledge, only a single study has actually applied Rep-PCR to type Lactobacillus isolates from chickens (62) for the identification of potential probiotics to control Salmonella enterica serovar Enteritidis in egg-laying hens.The aim of this study was to investigate whether Rep-PCR could be used to analyze Lactobacillus species and strains in the chicken GIT. Several Rep-PCR techniques [REP-, ERIC-, and (GTG)₅-PCR] were compared for the ability to type strains of several Lactobacillus species commonly isolated from the chicken GIT (L. crispatus, L. gallinarum, L. johnsonii, and L. reuteri). ERIC-PCR was able to simultaneously type isolates to the species and strain levels, and its strain differentiation ability was comparable with that of PFGE. ERIC-PCR was further applied to high-throughput analysis of a large number of isolates collected from chickens raised under NE and commercial conditions.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.     

Coculture Fermentations of Bifidobacterium Species and Bacteroides thetaiotaomicron Reveal a Mechanistic Insight into the Prebiotic Effect of Inulin-Type Fructans

Four bifidobacteria, each representing a cluster of strains with specific inulin-type-fructan degradation capacities, were grown in coculture fermentations with Bacteroides thetaiotaomicron LMG 11262, a strain able to metabolize both oligofructose and inulin. In a medium for colon bacteria with inulin as the sole added energy source, the ability of the bifidobacteria to compete for this substrate reflected phenotypical variation. Bifidobacterium breve Yakult, a strain that was not able to degrade oligofructose or inulin, was outcompeted by B. thetaiotaomicron LMG 11262. Bifidobacterium adolescentis LMG 10734, a strain that could degrade oligofructose (displaying a preferential breakdown mechanism) but that did not grow on inulin, managed to become competitive when oligofructose and short fractions of inulin started to accumulate in the fermentation medium. Bifidobacterium angulatum LMG 11039^T, a strain that was previously shown to degrade all oligofructose fractions simultaneously and to be able to partially break down inulin, was competitive from the beginning of the fermentation, consuming short fractions of inulin from the moment they appeared. Bifidobacterium longum LMG 11047, representing a cluster of bifidobacteria that shared both high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin, was the dominating strain in a coculture with B. thetaiotaomicron LMG 11262. These observations indicate that distinct subgroups within the large-intestinal Bifidobacterium population will be stimulated by different groups of prebiotic inulin-type fructans, a variation that could be reflected in differences concerning their health-promoting effects.The vast complexity of the human colon microbiota, the key element of the large-intestinal ecosystem, has inspired researchers to describe it as a postnatally acquired microbial organ located inside a host organ (1, 46). The microbial colon community is estimated to be composed of up to 100 trillion microorganisms, a number exceeding 10 times the total number of somatic and germ cells of a human adult (18, 38). The human microbiome is thought to contain more than 100 times the total number of human genes (1, 18). It not only broadens the digestive abilities of the host (18, 22, 40) but also influences body processes far beyond digestion (7, 33). In spite of its fundamental impact on human health and disease, the human gastrointestinal ecosystem remains largely unexplored (7, 8).Despite the fact that the present knowledge of the composition of the human large-intestinal microbiota is partial, fragmented, and undetailed, the consistency of some observations allows them to be generalized as facts (8, 28, 47). Notwithstanding the huge diversity at the strain level, up to 87% of the human colon inhabitants belong to only two bacterial phyla, the Bacteroidetes and the Firmicutes (1, 8, 14). Within the group of large-intestinal Bacteroidetes, large variations between individuals have been reported (8). However, Bacteroides spp. generally seem to account for up to 20% of the human colon microbiota (26, 32). Moreover, the presence of Bacteroides thetaiotomicron appears to be universal (8, 21). This species, which has been isolated only from human and rodent intestines or feces up to now, has gained importance as a perfect example of a flexible, niche-adapted, human symbiont with a wide carbohydrate consumption range (3, 4, 40).Although B. thetaiotaomicron is considered a human symbiont contributing to the stability of the colon ecosystem, the Bacteroides genus also harbors some notorious pathogens that are linked with severe extraintestinal infections and that have been mentioned as causal agents of acute diarrhea (30, 35). Moreover, besides their enormous saccharolytic potential, Bacteroides spp. are also capable of proteolytic fermentation (22). These considerations make them unsuited as target organisms for stimulation by prebiotics such as inulin-type fructans (23, 31).Most in vivo studies regarding the effect of the addition of inulin or oligofructose to the diet on the composition of the human colon microbiota reveal that Bacteroides spp. are neither stimulated nor repressed through administration of these prebiotics (34). However, at least some Bacteroides spp. are able to degrade inulin-type fructans, including B. thetaiotaomicron (13, 44). Since this species accounts for up to 6% of the colon microbiota (8), it is at least surprising that its numbers are hardly influenced by an increased availability of these prebiotics as substrates for large-intestinal fermentation. A possible explanation for these contradicting observations is to be found in the mechanism of inulin degradation, which in the case of Bacteroides is presumed to be periplasmic or even extracellular (37, 44). Leakage of free fructose toward the extracellular environment appears to be inherent in such breakdown mechanisms (10, 25, 44). Hence, extracellular fructan degraders inevitably provide opportunistic competitors, which are not able to degrade inulin-type fructans themselves, with a valuable source of energy (2, 10, 19). In contrast, a cell-associated or intracellular degradation mechanism is thought to be widespread among Bifidobacterium spp., which are still considered the main target organisms for prebiotic stimulation by inulin-type fructans (15, 16, 39, 44). This mechanism is often reflected in a clearly preferential breakdown of different-chain-length fractions of oligofructose, which approaches degradation of the long fractions only when short ones are depleted (10, 42, 44). The main disadvantage of such a cell-associated or intracellular degradation strategy seems to be the bifidobacterial incapacity to grow on long-chain-length fractions of inulin (36). Reports of the latter are indeed scarce: kinetic pure culture studies report an upper chain length limit for inulin degradation by Bifidobacterium spp., a disadvantage that will presumably not affect extracellular fructan degraders, such as Bacteroides spp. (9). Although the prebiotic effect of inulin-type fructans on the colon Bifidobacterium population is well documented, in vivo stimulation studies usually tend to consider the bifidobacterial community as a whole, ignoring interspecies differences (23). However, since the early days of in vitro prebiotic studies, a large variation in fructan degradation capacities of different Bifidobacterium strains has been reported (17, 36). It is likely that this variety is translated to the in vivo environment, implying that not all bifidobacteria are equally subject to prebiotic stimulation (5, 45). In a recent study, the kinetics of growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium spp., 17 of which were human intestinal isolates, have been statistically analyzed (9). The existence of four phenotypically distinct clusters among the tested strains, probably reflecting niche-specific adaptation, has been revealed. This rather limited variation was hypothesized to influence the susceptibilities of various bifidobacteria toward prebiotic stimulation by inulin-type fructans and their fitness to compete for these substrates in a complex environment, such as the colon ecosystem (44).The present study aimed at mapping the fructan degradation capacity of B. thetaiotaomicron LMG 11262 growing on oligofructose or inulin. In vitro competitiveness trials with bifidobacterial strains belonging to the different phenotypical clusters mentioned above were designed to investigate the abilities of these strains to compete for inulin in a coculture with an inulin-degrading B. thetaiotaomicron strain.     

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per μg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular serine proteases are one of the most important groups of industrial enzymes (13, 23). In addition, many extracellular metalloproteases play an important role in pathogenesis (36). Both types of proteases have been cloned and sequenced from many different individual microorganisms, i.e., Bacillus subtilis (56), Enterobacter sakazakii (22), Listeria monocytogenes (4), and Alteromonas sp. (33, 34).In the present study, we sought to isolate proteases by a metagenomic approach. Functional metagenomics comprising the isolation of DNA from environmental samples without prior enrichment of individual microorganisms, the construction of libraries from the recovered DNA, and function-driven screening of the generated libraries has led to the identification and characterization of a variety of novel enzymes (7, 14, 43), i.e., amylases (44, 58), oxidoreductases (21), lipolytic enzymes (9, 16, 44), monooxygenases (53), and proteins conferring nickel resistance (32). Despite the wealth of different biotechnologically important enzyme activities derived from metagenomes, little information on the characteristics of metagenome-derived proteases is available. One metagenome-derived fibrinolytic metalloprotease has been recovered (26). In addition, one serine protease has been cited in reviews (13, 29). In other metagenome projects, activity-based screening was unsuccessful (19, 44), or the thereby recovered proteolytic clones were not characterized (47).We report here on the identification and characterization of two metagenome-derived metalloproteases. We constructed metagenomic DNA libraries from environmental samples by a T4 DNA ligase-based and a topoisomerase-based cloning method. The suitability of both approaches for the construction of small-insert metagenomic libraries was compared The resulting library-containing Escherichia coli clones were screened for proteolytic activity. The plasmids were recovered from the positive E. coli strains and the insert sequences of two plasmids (pTW2 and pTW3), which conferred strong proteolytic activity, were sequenced. The targeted protease-encoding genes and the corresponding gene products were characterized.     

Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries (17, 32). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (30), because it can be converted into a variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (17, 32). Although succinic acid is currently produced from petroleum-derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged during the past decade (9, 10, 17).Several natural succinate-producing rumen bacteria that have high rates of succinate production and high succinate yields, such as Anaerobiospirillum succiniciproducens (22), Actinobacillus succinogenes (13, 28), and “Mannheimia succiniciproducens” (15, 16), have been isolated. However, these strains require complex organic nutrients that increase the costs associated with production, purification, and waste disposal (15, 22, 28). Low levels of succinate are produced by native strains of Escherichia coli in complex and mineral salts media (1, 4). Most mutant strains of E. coli that have been described previously as succinate producers also require complex organic nutrients (18, 23-26, 29, 31). Many involve two-step aerobic and anaerobic processes (3, 23-25, 29) and the addition of foreign genes (5, 6, 23-26, 29, 31).Novel E. coli biocatalysts (KJ060, KJ071, and KJ073) for the anaerobic production of succinate in mineral salts medium have been developed recently without the use of foreign genes or resident plasmids (9, 10). These biocatalysts were developed by combining constructed mutations to eliminate alternative routes of NADH oxidation in the mixed-acid pathway with growth-based selection (metabolic evolution). In subsequent studies (33), these strains were found to have recruited the glucose-repressed (7), gluconeogenic pck gene (11, 12, 19, 21, 27), encoding phosphoenolpyruvate carboxykinase (PCK) (derepressed via a point mutation in the promoter region), to replace the native phosphoenolpyruvate carboxylase (ppc) and serve as the primary route for CO₂ fixation (Fig. ​(Fig.1).1). A second acquired mutation was also identified as a frameshift mutation in the carboxy terminus of ptsI, inactivating the phosphoenolpyruvate-dependent phosphotransferase system (33). Glucose uptake by the phosphotransferase system was functionally replaced by galactose permease (galP) and glucokinase (glk).Open in a separate windowFIG. 1.Anaerobic metabolism of E. coli using the mixed-acid fermentation pathway (data from reference 1). The native phosphotransferase system pathway for glucose uptake and the mixed-acid pathway for fermentation are shown with black arrows. Peripheral reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown as dotted green arrows and represent new metabolic functions that have been recruited for succinate production from glucose. Reactions that have been blocked by gene deletions or point mutations are marked with an X. pck* indicates a novel mutation that derepressed pck, allowing the enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites but is presumed to exist as a single intracellular pool.Based on these previous studies, we have now determined the core mutations needed to direct carbon flow from glucose to succinate in E. coli and have constructed new succinate-producing strains with a minimum of genetic change.