Genome sequence of Soos: a siphovirus of the CP cluster infecting Gordonia rubripertincta  .

Novel actinobacteriophage Soos was isolated and purified from Southern Indiana soil using host Gordonia rubripertincta NRRL B-16540. Sequencing revealed a 57,509 bp circularly permuted genome encoding 87 predicted protein-coding genes. Soos is only the third phage in cluster CP, along with phages Clawz and Sting.

Genome Sequence of Gordonia rubripertincta Phage Survivors, a Cluster CT Siphovirus.

Bacteriophage Survivors is a siphovirus isolated from Gordonia rubripertincta NRRL B-16540. Survivors has a 45,436-bp genome encoding 69 predicted protein-coding genes, of which 32 have assigned functions. Based on gene content similarity to sequenced actinobacteriophages, Survivors is assigned to phage cluster CT.

Role of the carboxy terminus of SecA in iron acquisition, protein translocation, and virulence of the bacterial pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3' end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2'-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.

Cluster J mycobacteriophages: intron splicing in capsid and tail genes.

Bacteriophages isolated on Mycobacterium smegmatis mc(2)155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.

Iron acquisition functions expressed by the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium that causes serious infections in compromised patients. More recently, it has emerged as the causative agent of severe infections in military personnel wounded in Iraq and Afghanistan. This pathogen grows under a wide range of conditions including iron-limiting conditions imposed by natural and synthetic iron chelators. Initial studies using the type strain 19606 showed that the iron proficiency of this pathogen depends on the expression of the acinetobactin-mediated iron acquisition system. More recently, we have observed that hemin but not human hemoglobin serves as an iron source when 19606 isogenic derivatives affected in acinetobactin transport and biosynthesis were cultured under iron-limiting conditions. This finding is in agreement with the observation that the genome of the strain 17978 has a gene cluster coding for putative hemin-acquisition functions, which include genes coding for putative hemin utilization functions and a TonBExbBD energy transducing system. This system restored enterobactin biosynthesis in an E. coli ExbBD deficient strain but not when introduced into a TonB mutant. PCR and Southern blot analyses showed that this hemin-utilization gene cluster is also present in the 19606 strain. Analysis of the 17978 genome also showed that this strain harbors genes required for acinetobactin synthesis and transport as well as a gene cluster that could code for additional iron acquisition functions. This hypothesis is in agreement with the fact that the inactivation of the basD acinetobactin biosynthetic gene did not affect the growth of A. baumannii 17978 cells under iron-chelated conditions. Interestingly, this second iron uptake gene cluster is flanked by perfect inverted repeats and includes transposase genes that are expressed transcriptionally. Also interesting is the observation that this additional cluster could not be detected in the type strain 19606, an observation that suggests some significant differences in the iron uptake capacity between these two A. baumannii strains. Transposome mutagenesis of the strain 19606 resulted in the isolation of a derivative unable to grow under iron-chelated conditions. Gene mapping and protein analysis together with complementation assays showed that a protein related to SecA, which is a component of the Sec protein secretion system in a wide range of bacteria, is needed at least for the production of the BauA acinetobactin outer membrane receptor. Furthermore, this derivative was unable to use hemin as an iron source under limiting conditions. Taken together, these results indicate that A. baumannii expresses siderophore-mediated and hemin acquisition functions, although different isolates differ in their iron acquisition capacity. Unexpectedly, the ability of this pathogen to acquire iron depends on the expression of a SecA protein secretion function, which has not been associated with iron acquisition in bacteria.

Sec16 is a determinant of transitional ER organization.

BACKGROUND: Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization. RESULTS: We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks. CONCLUSIONS: Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization.

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.

The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.

The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related.

The Acinetobacter baumannii type strain, ATCC 19606, secretes acinetobactin, a catechol siderophore highly related to the iron chelator anguibactin produced by the fish pathogen Vibrio anguillarum (Listonella anguillarum). This paper reports the initial characterization of the genes and gene products involved in the acinetobactin-mediated iron-acquisition process. Insertional mutagenesis resulted in the isolation of several derivatives whose ability to grow in medium containing the iron chelator 2,2'-dipyridyl was affected. One of the insertions disrupted a gene encoding a predicted outer-membrane protein, named BauA, highly similar to FatA, the receptor for ferric anguibactin. Immunological relatedness of BauA with FatA was confirmed by Western blot analysis. Another transposon insertion was mapped to a gene encoding a protein highly similar to FatD, the permease component of the anguibactin transport system. Further DNA sequencing and nucleotide sequence analysis revealed that these A. baumannii 19606 genes are part of a polycistronic locus that contains the bauDCEBA ORFs. While the translation products of bauD, -C, -B and -A are highly related to the V. anguillarum FatDCBA iron-transport proteins, the product of bauE is related to the ATPase component of Gram-positive ATP-binding cassette (ABC) transport systems. This entire locus is flanked by genes encoding predicted proteins related to AngU and AngN, V. anguillarum proteins required for the biosynthesis of anguibactin. These protein similarities, as well as the structural similarity of anguibactin and acinetobactin, suggested that these two siderophores could be utilized by both bacterial strains, a possibility that was confirmed by siderophore utilization bioassays. Taken together, these results demonstrate that these pathogens, which cause serious infections in unrelated hosts, express very similar siderophore-mediated iron-acquisition systems.

The transitional ER localization mechanism of Pichia pastoris Sec12.

COPII vesicles assemble at ER subdomains called transitional ER (tER) sites, but the mechanism that generates tER sites is unknown. To study tER biogenesis, we analyzed the transmembrane protein Sec12, which initiates COPII vesicle formation. Sec12 is concentrated at discrete tER sites in the budding yeast Pichia pastoris. We find that P. pastoris Sec12 exchanges rapidly between tER sites and the general ER. The tER localization of Sec12 is saturable and is mediated by interaction of the Sec12 cytosolic domain with a partner component. This interaction apparently requires oligomerization of the Sec12 lumenal domain. Redistribution of P. pastoris Sec12 to the general ER does not perturb the localization of downstream tER components, suggesting that Sec12 and other COPII proteins associate with a tER scaffold. These results provide evidence that tER sites form by a network of dynamic associations at the cytosolic face of the ER.

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast.

The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/alpha3 subunit is deleted. Purified pre9Delta proteasomes show a two-fold enrichment for the Pre6/alpha4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/alpha4 is expressed in yeast, it complements not only pre6Delta but also pre6Delta pre9Delta mutants; therefore, the plant alpha4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Delta cells, suggesting an advantage for Pre9 over Pre6 incorporation at the alpha3 position that facilitates correct assembly.