| 1. |
Jian W,
Zhu L,
Dong X,
( 2001 ) New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences. PMID : 11594590 : DOI : 10.1099/00207713-51-5-1633 Abstract >>
The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows: 99.4-100% within the same species, 96% at the subspecies level, and 73-96% (mean 85%) at the interspecies level (and 98% in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 165 rRNA sequence similarities are all above 99%). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79% (mean 75%). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol% G+C) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-G+C (55-67 mol%) bifidobacteria were grouped into one cluster, whereas the low-G+C species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol%) and Bifidobacterium denticolens (55 mol%); a Bifidobacterium species of intermediate G+C content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.
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2. |
Ventura M,
Canchaya C,
Bernini V,
Del Casale A,
Dellaglio F,
Neviani E,
Fitzgerald GF,
van Sinderen D,
( 2005 ) Genetic characterization of the Bifidobacterium breve UCC 2003 hrcA locus. PMID : 16332909 : DOI : 10.1128/AEM.71.12.8998-9007.2005 PMC : PMC1317471 Abstract >>
The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of Bifidobacterium breve UCC 2003 revealed a second copy of a dnaJ gene, named dnaJ2, which is flanked by the hrcA gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial dnaJ sequences, including both dnaJ1 and dnaJ2 sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the B. breve UCC 2003 dnaJ2 gene seems to be regulated in a manner that is different from that of the previously characterized dnaJ1 gene. The dnaJ2 gene, which was shown to be part of a 2.3-kb bicistronic operon with hrcA, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized dnaJ genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.
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3. |
Yin X,
Chambers JR,
Barlow K,
Park AS,
Wheatcroft R,
( 2005 ) The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification. PMID : 15899413 : DOI : 10.1016/j.femsle.2005.04.013 Abstract >>
The nucleotide sequence of the xfp-gene region in six known and two unknown species of Bifidobacterium was determined and compared with the published sequences of B. animalis subsp. lactis DSM10140 and B. longum biovar longum NCC2705. The xfp coding sequences were 73% identical and coded for 825 amino acids in all 10 sequences. Partial sequences of an adjacent gene, guaA, were 61% identical in six sequences for which data were available. The region between xfp and guaA was variable in both length and sequence. Oligonucleotide sequences from the conserved and variable xfp regions were used as PCR primers, in combinations of appropriate specificity, for the detection and identification of Bifidobacterium isolates.
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4. |
Ventura M,
Zhang Z,
Cronin M,
Canchaya C,
Kenny JG,
Fitzgerald GF,
van Sinderen D,
( 2005 ) The ClgR protein regulates transcription of the clpP operon in Bifidobacterium breve UCC 2003. PMID : 16321946 : DOI : 10.1128/JB.187.24.8411-8426.2005 PMC : PMC1317013 Abstract >>
Five clp genes (clpC, clpB, clpP1, clpP2, and clpX), representing chaperone- and protease-encoding genes, were previously identified in Bifidobacterium breve UCC 2003. In the present study, we characterize the B. breve UCC 2003 clpP locus, which consists of two paralogous genes, designated clpP1 and clpP2, whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that the clpP1 and clpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of a clgR homologue, known to control the regulation of clpP gene expression in Streptomyces lividans and Corynebacterium glutamicum, was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of the clpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to the clpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.
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5. |
Ventura M,
Canchaya C,
Zink R,
Fitzgerald GF,
van Sinderen D,
( 2004 ) Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses. PMID : 15466567 : DOI : 10.1128/AEM.70.10.6197-6209.2004 PMC : PMC522111 Abstract >>
The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins. The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon. Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions. The heat inducibility of the groEL and groES genes of B. breve UCC 2003 was verified by slot blot analysis. Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit. The transcription initiation sites of these two mRNAs were determined by primer extension. Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones. Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently. Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker. On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus.
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