Endophytic and Biological Control Potential of Bacillus mojavensis and Related Species

The identity of a patented endophytic bacterium was established by 16S rRNA sequence analysis as a strain of Bacillus mojavensis, a recently erected species within one of the B. subtilis subgroups. This strain of B. mojavensis is antagonistic to the fungus Fusarium moniliforme, an endophytic mycotoxin-producing pathogen of maize and other plants. There are five other species within this subgroup: Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, Brevibacterium halotolerans, Paenibacillus lentimorbus, and P. popilliae. The objectives of this research were to screen other isolates of B. mojavensis, B. subtilis, and the other closely related Bacillus species for endophytic colonizing capacity and to determine the in vitro antagonism to F. moniliforme in an effort to survey the distribution of these traits, which are desirable biological control qualities within the Bacillaceae. Antagonism was determined on nutrient agar, and endophytic colonization was established with maize plants following recovery of rifampin-resistant mutants generated from all strains used in the study. The study established that all 13 strains of B. mojavensis, isolated from major deserts of the world, endophytically colonized maize and were antagonists to F. moniliforme. The endophytic colonization of maize by B. subtilis and other species within this subgroup of the Bacillaceae varied, as did antagonism, to F. moniliforme. Thus, this study suggests that endophytic colonization is another characteristic of the species B. mojavensis. The endophytic habit and demonstrated antagonism to the test fungus indicate that isolates of this species might prove to be important biological control organisms where the endophytic habit is desired.     

Design of a PCR test based on the gyrA gene sequence for the identification of closely related species of the Bacillus subtilis group

A method for the taxonomic identification of seven closely related bacterial species of the Bacillus subtilis group (B. subtilis, B. amyloliquefaciens, B. licheniformis, B. vallismortis, B. atrophaeus, B. sonorensis, and B. mojavensis) using specific primers selected on the basis of the gyrA gene sequences was developed. The effectiveness of this method both for the identification of pure cultures of type strains of this group and for the precise species identification of collection and industrial bacterial strains was demonstrated. The principal possibility of using this method for detecting B. subtilis group bacteria in mixed cultures was shown.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Relaxation of supercoiled DNA associated with induction of heat shock proteins in Escherichia coli

Bacillus subtilis 168 is known to possess two thymidylate synthase (TSase; EC 2.1.1.45) genes: thyA and thyB. thyB encodes a thermosensitive TSase (inactivated at 46° C) which, in wild-type cells, accounts for only 5–8% of the total cellular TSase activity. In order to investigate the thermal lability of TSaseB we have analyzed the thyB genes of B. subtilis 168 and of an unrelated strain B. subtilis ATCC6633, which is shown here to have a temperature-resistant TSaseB. This conclusion is supported by the frequency of appearance of spontaneous Thy^– mutants at 37° C and 46° C, and by the analysis of clones containing the thyB genes from the two strains. The nucleotide sequences of these two thyB genes were compared.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Structure of the Transposable Genetic Element ISBsu2 from the Cryptic Plasmid p1516 of a Soil Bacillus subtilis Strain and the Presence of Homologues of This Element in the Chromosomes of Various Bacillus subtilis Strains

A cryptic plasmid from a soil strain of Bacillus subtiliswas found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.     

Detailed identification of desert-originated bacteria carried by Asian dust storms to Japan

Several halotolerant bacteria were isolated from dust allowed to settle passively on saline medium in Higashi-Hiroshima, Japan during Asia dust events in 2005–2006. The primary identification, based on the sequence similarity of the 16S rRNA gene, revealed that these isolates were strains of Bacillus subtilis, B. licheniformis, Staphylococcus epidermidis, Gracillibacillus sp., and Halomonas venusta. A parallel investigation carried out on desert sand collected directly from sand dunes in Dunhuang, Gobi Desert, China resulted in the revivification of seven bacterial strains that were highly identical to the B. subtilis and B. licheniformis strains obtained in Higashi-Hiroshima (99.7 and 100% of 16S rDNA sequence similarity, respectively). A subsequent genetic analysis on the group of B. licheniformis isolates based on the universally house-keeping genes, gyrB and parE, revealed high sequence similarities in both genes among the strains of both locations (99.0–99.4%), which clustered them in a monophyletic line. Phenotype characterized by numerical taxonomy for 150 physiological tests confirmed the close relatedness between strains (similarity coefficient S _SM = 96.0%). The remarkable agreement between phenotype and genotype of the bacterial isolates allows us to conclude that there may have been an aerosolized dispersion of a Gobi Desert B. licheniformis by dust storms to Japan. This study provides evidence of microbial transport by yellow dust events in North-East Asia.     

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.     

Diversity of Aerobic Bacilli Analysis Using Molecular and Culture-Based Approaches in Debagh Hot Spring

This study reports the first attempt to describe aerobic bacilli communities in Debagh hot spring, from which 41 aerobic, thermophile, and halotolerant bacilli were isolated and selected based on morphological, physiological, and biochemical tests. 16S rDNA sequence analysis revealed that the recovered isolates belonged to four bacterial genera dominated by the genus Bacillus represented with species B. mojavensis (16), B. licheniformis (11), B. subtilis (2), B. atrophaeus (1), B.amyloliquifaciens (1), and B .pimulus (1). The genus Aeribacillus represented by the species A. pallidus (3), the genus Geobacillus represented by the species G. toebii (2), and the genus Hydrogenophilus represented by the species H. hirschii (4). While, MALDI-TOF analysis determined that isolates belonged to the genus Bacillus that contained B. licheniformis (12), B. mojavensis (6), B. subtilis (2), B. atrophaeus (1), and B. pumilus (1). Furthermore, the isolates exhibited high hydrolytic activity to casein, lecithin, tween 80, olive oil, and starch with 53.65%, 83.33%, 70.73%, 92.68%, and 56.09%, respectively. Among these isolates, 26.82% were able to hydrolyze all the substrates tested.     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various Bacillus Species

The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.     

关防风根腐病拮抗细菌筛选与鉴定

【背景】由木贼镰刀菌[Fusarium equiseti(Corda)Sacc.]引起的关防风根腐病,是近年来导致关防风产量及质量下降的主要土传病害之一。生物防治因其对环境安全及人畜无害等优势,成为目前植物病害防治的一种有效手段。【目的】挖掘关防风根际土壤中对木贼镰刀菌具有良好拮抗作用的生防菌株。【方法】采用稀释平板法分离根际土壤细菌;用滤纸片法和牛津杯法对拮抗细菌进行筛选和抑菌谱检测;用抗生素标记法标记拮抗细菌并测定其定殖能力;通过盆栽实验研究其对关防风根腐病的防效;通过形态学、生理生化特征及16S rRNA基因序列确定分类学地位。【结果】从健康关防风根际土壤中分离纯化了157株细菌,筛选获得对关防风根腐病菌具有显著抑菌作用的拮抗细菌SC-119,无菌滤液的抑菌率可达68.53%,而且兼具广谱抑菌能力和良好的定殖能力;盆栽实验表明,菌株SC-119对关防风根腐病的防治效果达到67.39%,其相对防效较接种哈茨木霉菌剂、枯草芽胞杆菌菌剂和代森锰锌分别提高了29.03%、32.26%和16.13%;对菌株SC-119进行分类学鉴定,确定其为萎缩芽胞杆菌(Bacillus atrophae...     

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.     

Cold-adapted Bacilli isolated from the Qinghai–Tibetan Plateau are able to promote plant growth in extreme environments

Nearly 1400 Bacillus strains growing in the plant rhizosphere were sampled from different sites on the Qinghai–Tibetan Plateau. Forty-five of the isolates, selected due to their biocontrol activity, were genome-sequenced and their taxonomic identification revealed that they were representatives of the Bacillus subtilis species complex (20) and the Bacillus cereus group (9). Majority of the remaining strains were found closely related to Bacillus pumilus, but their average nucleotide identity based on BLAST and electronic DNA/DNA hybridization values excluded closer taxonomic identification. A total of 45 different gene clusters involved in synthesis of secondary metabolites were detected by mining the genomes of the 45 selected strains. Except eight mesophilic strains, the 37 remaining strains were found either cold-adapted or psychrophilic, able to propagate at 10°C and below (Bacillus wiedmannii NMSL88 and Bacillus sp. RJGP41). Pot experiments performed at 10°C with winter wheat seedlings revealed that cold-adapted representatives of B. pumilus, B. safensis and B. atrophaeus promoted growth of the seedlings under cold conditions, suggesting that these bacilli isolated from a cold environment are promising candidates for developing of bioformulations useful for application in sustainable agriculture under environmental conditions unfavourable for the mesophilic bacteria presently in use.     

Wet and dry density of Bacillus anthracis and other Bacillus species

Aims: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. Methods and Results: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1·162 and 1·165 g ml^?1 and the other group included strains whose spores showed higher density values between 1·174 and 1·186 g ml^?1. Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R² = 0·95) between density and volume for the spores of all strains and species studied. Conclusions: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.     

枯草芽孢杆菌中高效响应N-乙酰神经氨酸生物传感器的构建

枯草芽孢杆菌(Bacillus subtilis)是公认的食品安全菌株,目前已被用于多种高附加值产品的生物合成,包括被广泛用作营养化学品和药物中间体的N-乙酰神经氨酸(N-acetylneuraminic acid, NeuAc)。响应目标产物的生物传感器被广泛用于代谢工程中的动态调控和高通量筛选等方面,以提高生物合成效率。但是,枯草芽孢杆菌中缺乏可高效响应NeuAc的生物传感器。因此,本文首先测试和优化了能将胞外NeuAc转运进胞内的转运蛋白,获得了一系列具有不同转运能力的菌株,以用于后续响应NeuAc的生物传感器的验证;随后将响应NeuAc的转录因子Bbr_NanR的结合位点插入枯草芽孢杆菌组成型启动子的不同位置,筛选具有活性的杂合启动子;接下来,通过在具有NeuAc转运能力的枯草芽孢杆菌中表达Bbr_NanR,选择能响应NeuAc的杂合启动子,并进一步通过优化Bbr_NanR表达量获得了一系列动态范围广、激活倍数高的生物传感器,其中生物传感器P535-N2能灵敏地响应胞内NeuAc浓度的变化,具有最大的动态范围,为(180–20 245) AU/OD;P566-N2则具有最高的激活倍数,为122倍,是已报道的枯草芽孢杆菌中响应N-乙酰神经氨酸的生物传感器的2倍。本文构建的响应NeuAc的生物传感器可用于高产NeuAc的酶突变体和枯草芽孢杆菌菌株的筛选,为枯草芽孢杆菌生物合成NeuAc提供了高效、灵敏的分析和调控工具。     

Sequence analysis of the Brevibacterium lactofermentum trp operon

Summary Brevibacterium lactofermentum, a Gram-positive bacterium, is a commercially important amino acid producer. In this organism, the tryptophan biosynthetic enzymes are encoded within a 7725 bp HapII-BamHI fragment. Seven open reading frames were identified as trp genes by complementation tests with various B. lactofermentum and Escherichia coli tryptophan auxotrophs. Following the nomenclature established for E. coli and Serratia marcescens, the B. lactofermentum trp genes were designated trpL, trpE, trpG, trpD, trpC (including the trpF domain), trpB, and trpA. The organization of these genes is identical to that in S. marcescens. The nucleotide sequences of the putative ribosome-binding sites for the B. lactofermentum trp genes resemble those of E. coli and Bacillus subtilis. Computer analysis revealed that the trp enzymes of B. lactofermentum resemble the enzymes of the Gram-negative E. coli more closely than those of the Gram-positive B. subtilis.Abbreviations bp base pairs - kb kilobases