Rapid and Reliable Identification of Food-Borne Yeasts by Fourier-Transform Infrared Spectroscopy

Computer-based Fourier-transform infrared spectroscopy (FT-IR) was used to identify food-borne, predominantly fermentative yeasts. Dried yeast suspensions provided the films suitable for FT-IR measurement. Informative windows in the spectrum were selected and combined to achieve optimal results. A reference spectrum library was assembled, based on 332 defined yeast strains from international yeast collections and our own isolates. All strains were identified with conventional methods using physiological and morphological characteristics. In order to assess identification quality, another 722 unknown yeast isolates not included in the reference spectrum library were identified both by classical methods and by comparison of their FT-IR spectra with those of the reference spectrum library. Ninety-seven and one-half percent of these isolates were identified correctly by FT-IR. Easy handling, rapid identification within 24 h when starting from a single colony, and a high differentiation capacity thus render FT-IR technology clearly superior to other routine methods for the identification of yeasts.Yeasts not only provided humans with the first biotechnologically produced food such as wine, bread, and fermented milk products but are also responsible for food spoilage (19), and some species are of medical importance. Therefore, a reliable method of yeast identification is economically significant (40). Furthermore, until now about 700 yeast species have been described. Since only a few habitats have been investigated in detail so far, a wide range of yeasts is likely to be discovered in the future (6). Exploration of new species includes the identification of a large number of isolates in order to eliminate duplicates and to discover unusual forms. For such tasks, a rapid, simple, low-cost identification method is needed. Conventional differentiation systems using morphological characters as well as patterns of the assimilation and fermentation of carbon sources (4, 22, 35) do not fulfil these requirements (9, 33, 38, 40). They are tedious and time-consuming, and, quite often, their capacity is limited since many species are distinguished from one another by a single physiological reaction which is often controlled by only one mutable marker (4, 20).Alternative methods such as fatty acid analysis (1, 31), electrophoretic karyotyping (10), restriction fragment length polymorphism, and DNA fingerprinting (26, 37) have already been evaluated (8). Restriction enzyme analysis of PCR-amplified rDNA (2), randomly amplified polymorphic DNA (3, 27), and nucleic acid hybridization with oligonucleotide probes (21, 24) have also been used. While some of these techniques do provide satisfactory results, molecular methods in general are still difficult to perform on a routine basis in laboratories of the food industry.Fourier-transform infrared (FT-IR) spectroscopy is used for the identification of substances in chemical analyses (14). The wavelength of infrared radiation ranges from 1 μm to 1 mm (32). In general, the wave number ν, the reciprocal of the wavelength, is used as a physical unit for FT-IR spectroscopy. Infrared radiation is divided into near (ν = 12,500 to 4,000 cm⁻¹), middle (ν = 4,000 to 200 cm⁻¹), and far (ν = 200 to 10 cm⁻¹) infrared. In this work, only the middle infrared section was used. FT-IR spectroscopy involves the observation of vibrations of molecules that are excited by an infrared beam. Molecules are able to absorb the energy of distinct light quanta and start a rocking or rotation movement. The FT-IR spectrum uses only vibrations that lead to a change in the dipole moment (14). An infrared spectrum represents a fingerprint which is characteristic for any chemical substance.The composition of biological material and, thus, of its FT-IR spectrum, is exceedingly complex, representing a characteristic fingerprint. Some years ago, Naumann and coworkers suggested identifying microorganisms by FT-IR spectroscopy (28–30). In principle, a reference spectrum library is assembled based on well-characterized strains and species. The FT-IR spectrum of any unidentified isolate is then measured under the same conditions as those used for the reference spectra and is compared to spectra in the reference spectrum library. If the library contains an identical or a very similar spectrum, an identification is possible. The success of the method is, therefore, directly dependent on the complexity of the reference spectrum library. The application of FT-IR spectroscopy has been reported for some species of the genera Lactobacillus (7), Actinomyces (15), Listeria (18), Streptococcus (13), and Clostridium (11). There are two reports which present preliminary data indicating that eukaryotic microorganisms such as yeasts may also be identified by FT-IR (17, 36). However, all these studies are based on a very limited number of species and isolates. For verification of the method only a few strains, which often were part of the reference spectrum library as well, were used. It was, therefore, still unclear whether FT-IR spectroscopy indeed was a competitive identification method.The aim of this study was to develop a standardized sample preparation procedure for yeasts (suitable for the normal laboratory), to select the most significant spectral windows for efficient identification, and to assemble a spectral reference library of sufficient complexity. Last, the identification of a great variety of unknown yeast isolates by FT-IR spectroscopy and conventional techniques had to be done in order to verify the method.     

Rapid species and strain differentiation of non-tubercoulous mycobacteria by Fourier-Transform Infrared microspectroscopy

Rapid identification of non-tuberculous mycobacteria (NTM) species is important in clinical laboratories to stipulate the appropriate therapy and to offer a comprehensive infection control. We applied Fourier-Transform Infrared microspectroscopy to evaluate, whether the most frequent species of NTM can be rapidly and uniformly identified by this method using microcolonies of NTM growing on solid nutrient agar plates. To establish a standardized protocol, the heterogeneity of cell growth within the microcolonies and the reproducibility of measuring the IR spectra from whole mycobacterial microcolonies were first studied. Hierarchical cluster analysis applied to spectra obtained by linear mapping across microcolony imprints from fast- and slow-growing NTM revealed only little spectral variance between the various microcolony zones. In parallel, when repetitive measurements were performed on independently grown whole single microcolonies with diameters of 80 and 140 mum, excellent reproducibility could be achieved, verifying that mycobacterial microcolonies are well suited for FT-IR-based identification. Twenty-eight different and well-defined strains, comprising the most frequent species of NTM isolated in clinical laboratories, were used to create a classification system based on FT-IR spectra from single microcolonies. Hierarchical cluster analysis allowed the assignment of all isolates measured in replicates to their correct species-specific clusters. Additionally, a clear separation of all strains into strain-specific sub-clusters was observed. These results demonstrate the potential of FT-IR microspectroscopy to rapidly differentiate NTM at the species and strain level. The data so far obtained suggest that an extended spectral database, containing more NTM strains and covering a broader biological variance, may provide a practical solution to rapidly identify unknown NTM isolates in routine clinical-microbiological laboratories with the additional possibility to type these microorganisms at the sub-species level.     

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2^−/− mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, Nos2^−/− mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.     

Synchrotron Fourier-Transform Infrared Microspectroscopy: Characterization of in vitro polarized tumor-associated macrophages stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells

Studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations accompanied by specific cellular and molecular mechanisms in the IBC tumor microenvironment (TME). IBC is characterized by significantly higher infiltration of tumor-associated macrophages (TAMs) that contribute to its metastatic process via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. Thus, there is a need to first understand how IBC-TME modulates the polarization of TAMs to better understand the role of TAMs in IBC. Herein, we used gene expression signature and Synchrotron Fourier-Transform Infrared Microspectroscopy (SR-μFTIR) to study the molecular and biochemical changes, respectively of in vitro polarized TAMs stimulated by the secretome of IBC and non-IBC cells. The gene expression signature showed significant differences in the macrophage's polarization-related genes between stimulated TAMs. FTIR spectra showed absorption bands in the region of 1700–1500 cm^?1 attributed to the amide I ν(C=O), & ν_AS (CN), δ (NH), and amide II ν(CN), δ (NH) proteins bands. Moreover, three peaks of different intensities and areas were detected in the lipid region of the νCH₂ and νCH₃ stretching modes positioned within the 3000–2800 cm^?1 range. The PCA analysis for the second derivative spectra of the amide regions discriminates between stimulated IBC and non-IBC TAMs. This study showed that IBC and non-IBC TMEs differentially modulate the polarization of TAMs and SR-μFTIR can determine these biochemical changes which will help to better understand the potential role of TAMs in IBC.     

Biological and Biomedical Applications of Synchrotron Infrared Microspectroscopy

The high brightness of synchrotron light,which is about three orders of magnitudegreater than a thermal source, has beenexploited in biological and biomedicalapplications of infrared microspectroscopy. The potential of this analytical tool isdocumented in this article in the study ofhuman tissue (hair and skin) and individualcells: biochemical and bio-structuralchanges based on corresponding functionalgroups have been identified and imaged withunprecedented spatial resolution. Thistechnique also provides a new tool foranalysis of biochemical kinetics of samplesduring disease and treatment. In the future, the combination ofinfrared microspectroscopy with othersynchrotron-based microscopic techniques,such as X-ray microscopy, at the samesample location is discussed.     

NSIT: Novel Sequence Identification Tool

Novel sequences are DNA sequences present in an individual''s genome but absent in the human reference assembly. They are predicted to be biologically important, both individual and population specific, and consistent with the known human migration paths. Recent works have shown that an average person harbors 2–5 Mb of such sequences and estimated that the human pan-genome contains as high as 19–40 Mb of novel sequences. To identify them in a de novo genome assembly, some existing sequence aligners have been used but no computational method has been specifically proposed for this task. In this work, we developed NSIT (Novel Sequence Identification Tool), a software that can accurately and efficiently identify novel sequences in an individual''s de novo whole genome assembly. We identified and characterized 1.1 Mb, 1.2 Mb, and 1.0 Mb of novel sequences in NA18507 (African), YH (Asian), and NA12878 (European) de novo genome assemblies, respectively. Our results show very high concordance with the previous work using the respective reference assembly. In addition, our results using the latest human reference assembly suggest that the amount of novel sequences per individual may not be as high as previously reported. We additionally developed a graphical viewer for comparisons of novel sequence contents. The viewer also helped in identifying sequence contamination; we found 130 kb of Epstein-Barr virus sequence in the previously published NA18507 novel sequences as well as 287 kb of zebrafish repeats in NA12878 de novo assembly. NSIT requires 2GB of RAM and 1.5–2 hrs on a commodity desktop. The program is applicable to input assemblies with varying contig/scaffold sizes, ranging from 100 bp to as high as 50 Mb. It works in both 32-bit and 64-bit systems and outperforms, by large margins, other fast sequence aligners previously applied to this task. To our knowledge, NSIT is the first software designed specifically for novel sequence identification in a de novo human genome assembly.     

Fourier-Transform Raman and Fourier-Transform Infrared Spectroscopy (An Investigation of Five Higher Plant Cell Walls and Their Components)

Infrared and Raman spectra of sequentially extracted primary cell walls and their pectic polymers were obtained from five angiosperm plants. Fourier-transform Raman spectrometry was shown to be a powerful tool for the investigation of primary cell-wall architecture at a molecular level, providing complementary information to that obtained by Fourier-transform infrared microspectroscopy. The use of an extraction procedure using imidazole instead of cyclohexane trans-1,2-N,N,N[prime],N[prime]-diaminotetraacetate allows the extension of the infrared spectral window for data interpretation from 1300 to 800 cm-1, to 2000 to 800 cm-1, and allows us to obtain Raman spectra from extracted cell-wall material. Wall constituents such as pectins, proteins, aromatic phenolics, cellulose, and hemicellulose have characteristic spectral features that can be used to identify and/or fingerprint these polymers without, in most cases, the need for any physical separation. The Gramineae (rice [Oryza sativa], polypogon [Polypogon fugax steud], and sweet corn [Zea mays]) are spectroscopically very different from the nongraminaceous monocotyledon (onion [Allium cepa]) and the dicotyledon (carrot [Daucus carota]); this reflects differences in chemical composition and cross-linking of the walls. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed.     

FusionPro,a Versatile Proteogenomic Tool for Identification of Novel Fusion Transcripts and Their Potential Translation Products in Cancer Cells,

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Highlights

• •FusionPro, a versatile tool for studying fusion proteoforms, has been developed.
• •Fusion peptides were identified against a customized database built by FusionPro.
• •Types and features of fusion proteoforms were efficiently predicted by FusionPro.

     

Evaluation and Reliability of a Simplified Method for Identification of Food-Borne Yeasts

A simplified identification key described by Deak and Beuchat (T. Deak and L. R. Beuchat, J. Food Prot. 50:243-264, 1987) and the computer method of Barnett et al. (J. A. Barnett, R. W. Payne, and D. Yarrow, Yeast Identification Program, 1985) were used to identify 12 reference strains and 382 yeasts isolated from cultured milk products. Because the simplified key failed to account for species variability with regard to physiological, morphological, and sexual reproduction characteristics, poor agreement of the identification results was obtained. A reevaluation of the basic theoretical assumptions of the simplified key only confirmed the practical results and indicates that this identification method is unsatisfactory     

Discrimination Between Paraffin-Embedded and Frozen Skin Sections Using Synchrotron Infrared Microspectroscopy

The difference between paraffin-embedded and frozen skin sections is always questionable. Ten patients of early stage mycosis fungoides, ten patients with psoriasis and ten normal controls were included in this study. Aim of this study is to differentiate between paraffin-embedded and frozen skin sections in inflammatory and malignant dermatoses using synchrotron infrared microspectroscopy (SIRM). It was found that epidermal beta sheets in paraffin-embedded sections were higher in a highly significant manner than frozen sections (P < 0.001). Also, epidermal nucleic acids in paraffin-embedded sections were lower in a highly significant manner than frozen sections (P < 0.001). However, when various skin diseases were compared with the control. It was found that the difference between paraffin-embedded and frozen skin sections were almost similar. In conclusion SIRM is a unique promising diagnostic technique and it seems that frozen processing preserve skin tissue more, this was represented by less apoptosis (beta sheets) and more nucleic acids than paraffin processing. However, there are still many advantages of both approaches over the other depending on the goal of the study.     

Determination of Glucose Concentration in Whole Blood using Fourier-Transform Infrared Spectroscopy

Fourier-transform infrared(FTIR) transmission spectroscopy has beenused for the determination of glucoseconcentrations in whole blood samples fromtwenty-eight patients. A four-vectorpartial least squares calibration model,using the spectral range 950–1200 cm^-1,yielded a standard error of prediction of0.59 mM for an independent test set. Forblood samples from a single patient, wefound that the glucose concentration wasproportional to the difference between thevalues of the second derivative spectrum at1082 cm^-1 and 1093 cm^-1, suggestingthat these two specific wavelengths can beused for determining glucose concentrationsin blood.     

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra‐peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro‐spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis.

Intra‐peritoneal infection with high dose of Salmonella Typhimurium leads to rapid increase in inflammatory cytokines, e.g. Tnfα (A). FTIR analysis of liver (B) and sera (C) identifies several metabolic changes: glycogen, protein/lipid, cholesteryl esters and DNA.     

Amide 1 Expression in Psoriasis and Lichen Planus using Synchrotron Infrared Microspectroscopy

Psoriasis vulgaris and, Lichen planus are cutaneous inflammatory conditions that usually exhibit distinctive morphology. Ten psoriasis vulgaris and, ten Lichen planus patients (mean age, 45 ± 10.27 years) with confirmed histopathological diagnoses were analyzed. In the current study synchrotron infrared (IR) microspectroscopy was used to differentiate between these two conditions based on their lymphocytic proteins analyses. It was found that β-sheets protein structure, known to represent cell apoptosis, were expressed significantly in Lichen planus conditions than that of the psoriasis vulgaris when analyzed against the established normal control groups of five patients of comparable age and, genders (P = 0.001, 0.03 respectively). Also, the amide 1 protein type within the epidermis of Lichen planus were expressed in significant proportions as compared to psoriasis vulgaris (P < 0.001). On the contrary, the amide 1 protein structural types were found clustered in psoriasis vulgaris in different IR spectra than that in Lichen planus as observed in a number of patients during this study. These observations indicated that the concentration of amide 1 protein in psoriasis vulgaris varies to that of Lichen planus. In conclusion, both psoriasis vulgaris and, Lichen planus have different types of epidermal and, dermal protein structures and, this information can be of clinical diagnostic and therapeutic use for these cutaneous inflammatory conditions in near future.     

MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix‐assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI‐IMS MSI) to investigate amyloid deposits in formalin‐fixed and paraffin‐embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI‐IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample‐consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.     

Extremely Rapid Extraction of DNA from Bacteria and Yeasts

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.     

Resistance Gene Analogues as a Tool for Rapid Identification and Cloning of Disease Resistance Genes in Plants 3 A Review

Most of the disease resistance genes (R-genes) discovered in plants have conserved functional domains, predominantly among them are nucleotide binding sites (NBS) and leucine rich repeats (LRR). The sequence information of the conserved domains can be invariably used to mine similar sequences from other plant species, using degenerate and specific primers for their amplification in a polymerase chain reaction. Such derived sequences, known as Resistance Gene Analogues (RGAs), can serve as molecular markers for rapid identification and isolation of R-genes. Besides, they can also provide clues about the evolutionary mechanism of resistance genes and the interaction involved in pathogen recognition. In the recent years, this sequence-homology based approach has been used extensively for the cloning and mapping of RGAs in cereals, pulses, oilseeds, coffee, spices, forest trees and horticultural crops. In this article, the current status of cloning of RGAs from different crops has been reviewed. A general method of RGA cloning and its modifications like NBS-profiling and AFLP-NBS have also been discussed along with examples. Further, it has been suggested that the RGAs cloned in various crops would be a useful genomic resource for developing cultivars with durable resistance to diseases in different crop breeding programmes.     

糖苷合成酶——— 一类新型的寡糖高效合成工具

寡糖是哺乳动物细胞表面糖蛋白和糖脂以及微生物来源的生理活性物质的要素之一,其应用于医药的巨大潜能至今还没有得到充分体现,主要原因是合成足够于临床使用的寡糖非常困难.传统的化学法和酶法在大规模合成寡糖方面都有一定局限性.近年来,分子生物学技术大大推动了糖苷酶合成寡糖的研究,将糖苷酶催化中心亲核体氨基酸定点突变为非亲核体氨基酸,导致酶的原有水解活性丧失,只催化糖苷键合成反应,寡糖产量最高可达99%,人工产生了一类新酶——糖苷合成酶(glycosynthases),随后又产生了硫代糖苷酶(thioglycoligases)和硫代糖苷合成酶(thioglycosynthases).糖苷合成酶的高通量筛选可用双质粒系统和酵母三杂交系统进行,其活性的进一步改进可通过亲核体氨基酸位点不同氨基酸取代、其他位点氨基酸突变、反应条件优化等方法进行,其区域选择性的改变或增强可通过改变糖基受体分子达到.糖苷合成酶作为一种新型高效的生物催化剂,对寡糖的工业化合成有着重要意义,它的出现对糖生物学的发展必将起到巨大的推动作用.     

Near Infrared Spectroscopy Facilitates Rapid Identification of Both Young and Mature Amazonian Tree Species

Precise identification of plant species requires a high level of knowledge by taxonomists and presence of reproductive material. This represents a major limitation for those working with seedlings and juveniles, which differ morphologically from adults and do not bear reproductive structures. Near-infrared spectroscopy (FT-NIR) has previously been shown to be effective in species discrimination of adult plants, so if young and adults have a similar spectral signature, discriminant functions based on FT-NIR spectra of adults can be used to identify leaves from young plants. We tested this with a sample of 419 plants in 13 Amazonian species from the genera Protium and Crepidospermum (Burseraceae). We obtained 12 spectral readings per plant, from adaxial and abaxial surfaces of dried leaves, and compared the rate of correct predictions of species with discriminant functions for different combinations of readings. We showed that the best models for predicting species in early developmental stages are those containing spectral data from both young and adult plants (98% correct predictions of external samples), but even using only adult spectra it is still possible to attain good levels of identification of young. We obtained an average of 75% correct identifications of young plants by discriminant equations based only on adults, when the most informative wavelengths were selected. Most species were accurately predicted (75–100% correct identifications), and only three had poor predictions (27–60%). These results were obtained despite the fact that spectra of young individuals were distinct from those of adults when species were analyzed individually. We concluded that FT-NIR has a high potential in the identification of species even at different ontogenetic stages, and that young plants can be identified based on spectra of adults with reasonable confidence.     

Repetitive,Marker-Free,Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB_min), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB_min site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps.