Raul J. Cano

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.

Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus

Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter–operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract.

Identification and Inactivation of Genetic Loci Involved with Lactobacillus acidophilus Acid Tolerance

ABSTRACT Amino acid decarboxylation-antiporter reactions are one of the most important systems for maintaining intracellular pH between physiological limits under acid stress. We analyzed the Lactobacillus acidophilus NCFM complete genome sequence and selected four open reading frames with similarities to genes involved with decarboxylation reactions involved in acid tolerance in several microorganisms. Putative genes encoding an ornithine decarboxylase, an amino acid permease, a glutamate γ-aminobutyrate antiporter, and a transcriptional regulator were disrupted by insertional inactivation. The ability of L. acidophilus to survive low-pH conditions, such as those encountered in the stomach or fermented dairy foods, was investigated and compared to the abilities of early- and late-stationary-phase cells of the mutants by challenging them with a variety of acidic conditions. All of the integrants were more sensitive to low pH than the parental strain. Interestingly, each integrant also exhibited an adaptive acid response during logarithmic growth, indicating that multiple mechanisms are present and orchestrated in L. acidophilus in response to acid challenge.

Staphylococcus succinus sp. nov., isolated from Dominican amber

Two bacterial isolates, designated AMG-D1T and AMG-D2, were recovered from 25-35-million-year-old Dominican amber. AMG-D1T and AMG-D2 biochemically most closely resemble Staphylococcus xylosus; they differ physiologically from other staphylococci. Fatty acid analysis and comparisons with extensive databases were unable to show relatedness to any specific taxon. Moreover, AMG-D1T and AMG-D2 contain tuberculostearic acid and meso-diaminopimelic acid, characteristic of the G + C-rich coryneform bacteria, as opposed to L-lysine characteristic of staphylococci. AMG-D1T and AMG-D2 have a G + C ratio of 35 mol%. Phylogenetic analysis with the 16S rRNA gene indicated that AMG-D1T and AMG-D2 were most closely related to Staphylococcus equorum, S. xylosus, Staphylococcus saprophyticus and other novobiocin-resistant staphylococci. Stringent DNA-DNA hybridization studies with AMG-D1T revealed similarities of 38% with S. equorum, 23% with S. xylosus and 6% with S. saprophyticus. The results indicate that AMG-D1T and AMG-D2 represent a novel species, which was named Staphylococcus succinus sp. nov. The type strain of the new species is AMG-D1 (ATCC 700337).

Genetic Analysis of Two Bile Salt Hydrolase Activities in Lactobacillus acidophilus NCFM

ABSTRACT Two genes, bshA and bshB, encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.

Micrococcus luteus - Survival in Amber

A growing body of evidence now supports the isolation of microorganisms from ancient materials. However, questions about the stringency of extraction methods and the genetic relatedness of isolated organisms to their closest living relatives continue to challenge the authenticity of these ancient life forms. Previous studies have successfully isolated a number of spore-forming bacteria from organic and inorganic deposits of considerable age whose survival is explained by their ability to enter suspended animation for extended periods of time. However, despite a number of putative reports, the isolation of non-spore-forming bacteria and an explanation for their survival have remained enigmatic. Here we describe the isolation of non-spore-forming cocci from a 120-million-year-old block of amber, which by genetic, morphological, and biochemical analyses are identified as belonging to the bacterial species Micrococcus luteus. Although comparison of 16S rRNA sequences from the ancient isolates with their modern counterparts is unable to confirm the precise age of these bacteria, we demonstrate, using complementary molecular and cell biological techniques, evidence supporting the view that these (and related modern members of the genus) have numerous adaptations for survival in extreme, nutrient-poor environments, traits that will assist in this bacteria’s persistence and dispersal in the environment. The bacteria’s ability to utilize succinic acid and process terpine-related compounds, both major components of natural amber, support its survival in this oligotrophic environment.

Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa

Bacteriophage B3 is a transposable phage of Pseudomonas aeruginosa. In this report, we present the complete DNA sequence and annotation of the B3 genome. DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3%. The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons. Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins. Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes. Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions. Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster. These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.

Diversity of Microorganisms Isolated from Amber

A bstractClaims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25–40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice.

Sequence analysis of bacterial DNA in the colon of an Andean mummy.

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.