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BACKGROUND AND AIMS: The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. METHODS: Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. RESULTS: IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). CONCLUSIONS: IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

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AIMS: The objective of this study was to generate strain-specific genomic patterns of a bank of 67 commercial and reference probiotic strains, with a focus on probiotic lactobacilli. METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) was used as the primary method for strain differentiation. This method was compared with carbohydrate fermentation analysis. To supplement visual comparison, PFGE patterns were analysed quantitatively by cluster analysis using unweighted pair group method with arithmetic averages. SmaI, NotI and XbaI were found to effectively generate clear and easy-to-interpret PFGE patterns of a range of probiotic strains. Some probiotic strains from different sources shared highly similar PFGE patterns. CONCLUSIONS: Results document the value of genotypic strain identification methods, combined with phenotypic methods, for determining probiotic strain identity and relatedness. No correlation was found between relatedness determined by carbohydrate fermentation profiles alone compared with PFGE analysis alone. Some commercial strains are probably derived from similar sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is valuable to the probiotic industry to develop commercial strain identification patterns, to provide quality control of strain manufacturing production runs, to track use of protected strains and to determine the relatedness among different research and commercial probiotic strains.

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Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.

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Assessment of fungal diversity in environmental samples is currently a challenge. Several recently developed molecular methods offer new avenues for determining the presence and diversity of fungi in complex microbial communities. Terminal restriction fragment (TRF) pattern analysis was tested as a method for assessing the fungal molecular diversity of a terrestrial microbial community. Community DNA was isolated from sand samples taken from a pilot-scale petroleum-contaminated land treatment unit. PCR amplification was carried out using primers, one of which was fluorescently labeled, designed to hybridize to conserved sequences in the fungal ribosomal small subunit (18S) or the internal transcribed spacer ITS1-5.8S-ITS2 (ITS) ribosomal region. Amplicons were then digested separately with HpaII or HaeIII; fluorescently labeled TRFs were detected by capillary gel electrophoresis. ITS region TRF patterns were predicted and observed to generate a greater richness than 18S TRF patterns. Unique TRF patterns were also observed for each community examined. Finally, the ITS region showed a higher degree of specificity in matching observed TRF profiles to those generated from GenBank sequence data for species identification. These data suggest that ITS rDNA TRF pattern analysis has great potential as a rapid and specific method for fungal community analysis and species identification.

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Terminal Restriction Fragment (TRF) patterns, also known as Terminal Restriction Fragment Length Polymorphisms (T-RFLP), are a recently introduced PCR-based tool for studying microbial community structure and dynamics. Since the first review of TRF methodology (Marsh, 1999. Curr. Op. Microbiol. 2: 323-7), at least 35 new research articles were published that include this powerful tool in some part of their reports. This review covers some of the applications that TRF patterns were used for and provides a discussion of how to create and analyze TRF pattern data. This data has the advantage of being simply and rapidly produced using standard DNA sequencing equipment. The raw data are automatically converted to a digitized form that can be easily analyzed with a variety of multivariate statistical techniques. The identification of specific elements in a TRF pattern is possible by comparison to entries in a good sequence database or by comparison to a clone library. As an added advantage when investigating complex microbial communities such as those in soils and intestines, TRF patterns are recognized as having better resolution than other DNA-based methods for evaluating community structure.

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16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of Lactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence of L. johnsonii was confirmed by isolation from feces.

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Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.

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Biotransformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in slurry reactors was studied to determine the importance of supplementation of known biodegraders and the type of nutrient source required. Although addition of bacteria to the system increased the biotransformation rates, the increase may not justify the additional work and cost needed to grow the organisms in a laboratory and mix them into the soil. An inexpensive, rich nutrient source, corn steep liquor, was shown to provide sufficient nutrients to allow for the cometabolic biotransformation of RDX. The rate of RDX transformation was not constant throughout the course of the experiment due to the heterogeneous microbial population. Three kinetically distinct phases were observed. Regardless of the process, RDX biotransformation in slurry reactors was reaction rate limited under the test conditions. Model simulations based on experimental results demonstrate that, at cell densities of 5 g/L, bioremediation of RDX-contaminated soil is an attractive clean-up alternative.

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Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

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Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-source. This isolate, Pseudomonas aeruginosa MA01, metabolized TNT by first reducing one nitro group to form either 2-amino-4,6-dinitrotoluene (2ADNT) or 4-amino-2,6-dinitrotoluene (4ADNT). However, strain MA01 was distinct from other TNT-reducing organisms in that it transformed these compounds into highly polar metabolites through an O2-dependent process. Strain MA01 was able to cometabolize TNT, 2ADNT, and 4ADNT in the presence of a variety of carbon and energy sources. During aerobic cometabolism with succinate, 45% of uniformly ring-labeled [14C]TNT was transformed to highly polar compounds. Aerobic cometabolism of purified [14C]2ADNT and [14C]4ADNT with succinate as C-source produced similar amounts of these polar metabolites. During O2-limited cometabolism with succinate as C-source and nitrate as electron acceptor, less than 8% of the [14C]TNT was transformed to polar metabolites. Purified 2,6-diamino-4-nitrotoluene was not metabolized, and while 2,4-diamino-6-nitrotoluene was acetylated, the product (N-acetyl-2,4-diamino-6-nitrotoluene) was not further metabolized. Therefore, strain MA01 metabolized TNT by oxidation of the ADNTs and not by reduction the remaining nitro groups on the ADNTs.

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Three species of the family Enterobacteriaceae that biochemically reduced hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were isolated from nitramine explosive-contaminated soil. Two isolates, identified as Morganella morganii and Providencia rettgeri, completely transformed both RDX and the nitroso-RDX reduction intermediates. The third isolate, identified as Citrobacter freundii, partially transformed RDX and generated high concentrations of nitroso-RDX intermediates. All three isolates produced 14CO2 from labeled RDX under O2-depleted culture conditions. While all three isolates transformed HMX, only M. morganii transformed HMX in the presence of RDX.

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In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to O2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory e- donors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70% lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e- donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allow Azorhizobium spp. to flourish in wide-ranging O2 environments.

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A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.

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In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2. From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.

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