Genome Sequence and Characterization of Acinetobacter Phage DMU1.

Background: There has been a recent resurgence of research on the characterization of Acinetobacter phage for therapeutic use due to the morbidity and mortality associated with treatment failures in cases of multidrug-resistant Acinetobacter baumannii infections. Materials and Methods: A bacteriophage isolated from activated sludge that targets A. baumannii ATCC19606 was characterized by electron microscopy, genome sequencing, comparative genomics, and a host range analysis. Results: The morphology of Acinetobacter phage DMU1 resembles phages in Siphoviridae. Comparative genomic and phylogenetic analyses reveal that DMU1 is a siphophage and is most closely related to Acinetobacter phage SH-Ab 15497. Out of the strains tested, DMU1 was found to only infect A. baumannii strains ATCC19606 and ATCC17978. Conclusion: Acinetobacter phage DMU1 belongs to the Siphoviridae family and is most closely related to Acinetobacter phage SH-Ab 15497. Small-scale host-range analysis of DMU1 indicates a host range that is likely limited to specific A. baumannii strains.

Draft Genome Sequence of the Clinical Isolate Acinetobacter nosocomialis Strain M2.

We report the 3.78-Mbp high-quality draft assembly of the genome from a clinical isolate of Acinetobacter nosocomialis called strain M2 (previously known as Acinetobacter baumannii strain M2).

Analysis of autoinducer-2 quorum sensing in Yersinia pestis.

The autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system in Yersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in a luxS-dependent manner, reaching maximal levels of 2.5 µM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomal lsr locus encoding factors involved in the binding and import of AI-2, and confirming this assignment, an lsr deletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing in Y. pestis, microarray studies were conducted by comparing Δpgm strain R88 to a Δpgm ΔluxS mutant or a quorum-sensing-null Δpgm ΔypeIR ΔyspIR ΔluxS mutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may help Y. pestis survive at the host temperature. This was confirmed by observing that the luxS mutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling in Y. pestis and identifies potential roles for the system in controlling genes important to disease.

Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility.

UNLABELLED: Acinetobacter baumannii is a Gram-negative, opportunistic pathogen. Recently, multiple A. baumannii genomes have been sequenced; these data have led to the identification of many genes predicted to encode proteins required for the biogenesis of type IV pili (TFP). However, there is no experimental evidence demonstrating that A. baumannii strains actually produce functional TFP. Here, we demonstrated that A. baumannii strain M2 is naturally transformable and capable of twitching motility, two classical TFP-associated phenotypes. Strains were constructed with mutations in pilA, pilD, and pilT, genes whose products have been well characterized in other systems. These mutants were no longer naturally transformable and did not exhibit twitching motility. These TFP-associated phenotypes were restored when these mutations were complemented. More PilA was detected on the surface of the pilT mutant than the parental strain, and TFP were visualized on the pilT mutant by transmission electron microscopy. Thus, A. baumannii produces functional TFP and utilizes TFP for both natural transformation and twitching motility. Several investigators have hypothesized that TFP might be responsible, in part, for the flagellum-independent surface-associated motility exhibited by many A. baumannii clinical isolates. We demonstrated that surface-associated motility was not dependent on the products of the pilA, pilD, and pilT genes and, by correlation, TFP. The identification of functional TFP in A. baumannii lays the foundation for future work determining the role of TFP in models of virulence that partially recapitulate human disease. IMPORTANCE: Several investigators have documented the presence of genes predicted to encode proteins required for the biogenesis of TFP in many A. baumannii genomes. Furthermore, some have speculated that TFP may play a role in the unique surface-associated motility phenotype exhibited by many A. baumannii clinical isolates, yet there has been no experimental evidence to prove this. Unfortunately, progress in understanding the biology and virulence of A. baumannii has been slowed by the difficulty of constructing and complementing mutations in this species. Strain M2, a recently characterized clinical isolate, is amenable to genetic manipulation. We have established a reproducible system for the generation of marked and/or unmarked mutations using a modified recombineering strategy as well as a genetic complementation system utilizing a modified mini-Tn7 element in strain M2. Using this strategy, we demonstrated that strain M2 produces TFP and that TFP are not required for surface-associated motility exhibited by strain M2.

Acinetobacter baumannii utilizes a type VI secretion system for bacterial competition.

Type VI secretion systems (T6SS) are a class of macromolecular secretion machines that are utilized by a number of bacteria for inter-bacterial competition or to elicit responses in eukaryotic cells. Acinetobacter baumannii is an opportunistic pathogen that causes severe infections in humans. These infections, including pneumonia and bacteremia, are important, as they are often associated with hospitals and medical-settings where they disproportionally affect critically ill patients like those residing in intensive care units. While it is known that A. baumannii genomes carry genes whose predicted products have homology with T6SS-associated gene products from other bacteria, and secretion of a major T6SS structural protein Hcp has been demonstrated, no additional work on an A. baumannii T6SS has been reported. Herein, we demonstrated that A. baumannii strain M2 secretes Hcp and this secretion was dependent upon TssB, an ortholog of a bacteriophage contractile sheath protein, confirming that strain M2 produces a functional T6SS. Additionally, we demonstrated that the ability of strain M2 to out-compete Escherichia coli was reliant upon the products of tssB and hcp. Collectively, our data have provided the first evidence demonstrating function in inter-bacterial competition, for a T6SS produced by A. baumannii.

Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons.

We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.

Exploring the response of Escherichia coli O157:H7 EDL933 within Acanthamoeba castellanii by genome-wide transcriptional profiling.

Free-living protozoa, such as Acanthamoeba castellanii, are environmental hosts for pathogenic bacteria. Protozoa have been implicated in harboring pathogenic bacteria and enhancing virulence factors and antibiotic resistance. To better understand this relationship with Escherichia coli O157:H7, we characterized its transcriptome within A. castellanii compared with broth-grown organisms using two-color microarrays. Statistical analysis indicated that 969 genes were differentially expressed at P<0.018, with a false discovery rate of 1.9% and a fold change cutoff of 1.3 or greater. There were 655 upregulated transcripts that include 40 genes associated with virulence, of which 32 are encoded on O-islands, and include shiga toxin genes (stx1A, stx1B stx2A) and 14 genes involved in Type III secretion system components. Also included are SOS response genes such as lexA and recA, genes involved in or predicted to be involved in antibiotic resistance (rarD, macAB, marABR, mdtK, yojI, yhgN), the quorum-sensing operon lsrACDB, and the efe and feo iron-acquisition systems. There were 314 downregulated transcripts that included 19 transcripts associated with virulence, seven of which are encoded on O-islands. Our results demonstrate that a significant portion of the E. coli O157:H7 genome was differentially expressed as a result of the protozoan intracellular environment.

Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock.

The response to elevated temperature [heat shock (HS)] is highly conserved. The transcriptome of Escherichia coli K-12 has been studied under a variety of conditions while such studies involving E. coli O157:H7 are only now being conducted. To better understand the impact of HS on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37 to 50 degrees C for 15 min were compared with cells held at 37 degrees C by microarray. Using a mixed model analysis, 193 genes were found to be differentially transcribed at P<0.0042 with a q value <0.1. The 111 downregulated genes include the curli pili-associated genes csgABCDEFG, maltose transport-associated proteins malEFK, and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes upregulated include the HS-induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with two LEE-encoded genes: hypothetical gene Z5121 and sepZ. Twenty-three additional genes located in O-islands were found to be differentially expressed. Quantitative real-time PCR (qRT-PCR) was performed to validate the microarray results. Also, samples subjected to a 30-42 degrees C shift were examined by qRT-PCR to confirm differential transcription of selected genes. These results indicate that this pathogen may regulate its virulence factors in response to temperature changes.

Transcriptome analysis of organisms with food safety relevance.

Transcriptome analysis using microarrays has become a powerful tool to better understand the process of disease and other complex biological processes such as food spoilage and biofilm formation. This review is divided into two basic sections: 1) a short history and description of microarrays and 2) a discussion of studies involving bacterial food safety pathogens that focused on whole genome transcript analysis. Not included are the many studies using microarrays to identify, diagnose, or genetically characterize these organisms. This review focuses on studies involving Escherichia coli O157:H7, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. Many of the studies involve altering the growth environment to simulate stress conditions and the use of host-pathogen model systems to explore virulence mechanisms. Few studies use conditions that might be considered unique to the food industry. Exceptions are studies of biofilm-specific transcriptome changes and analysis following pressure treatment. This review should not be considered as a comprehensive review, and where appropriate, species-specific reviews are cited that are more complete.

Transcriptome changes in Mycoplasma hyopneumoniae during infection.

Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNA-enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2.7%. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Four down-regulated genes and two up-regulated genes encoded putative lipoproteins. secA (mhp295) (P = 0.003) and two glycerol transport permease genes (potA [mhp380; P = 0.006] and ugpA [mhp381; P = 0.003]) were up-regulated in vivo. Elongation factor EF-G (fusA [mhp083]) (P = 0.002), RNA polymerase beta chain (rpoC [mhp635]) (P = 0.003), adenylate kinase (adk [mhp208]) (P = 0.001), prolyl aminoacyl tRNA synthetase (proS [mhp397]) (P = 0.009), and cysteinyl-tRNA synthetase (cysS [mhp661]) (P < 0.001) were down-regulated in vivo.