The nucleoid protein HU positively regulates the expression of type VI secretion systems in Enterobacter cloacae.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

The interaction of InvF-RNAP is mediated by the chaperone SicA in Salmonella sp: an in silico prediction.

In this work we carried out an in silico analysis to understand the interaction between InvF-SicA and RNAP in the bacterium Salmonella Typhimurium strain LT2. Structural analysis of InvF allowed the identification of three possible potential cavities for interaction with SicA. This interaction could occur with the structural motif known as tetratricopeptide repeat (TPR) 1 and 2 in the two cavities located in the interface of the InvF and α-CTD of RNAP. Indeed, molecular dynamics simulations showed that SicA stabilizes the Helix-turn-Helix DNA-binding motifs, i.e., maintaining their proper conformation, mainly in the DNA Binding Domain (DBD). Finally, to evaluate the role of amino acids that contribute to protein-protein affinity, an alanine scanning mutagenesis approach, indicated that R177 and R181, located in the DBD motif, caused the greatest changes in binding affinity with α-CTD, suggesting a central role in the stabilization of the complex. However, it seems that the N-terminal region also plays a key role in the protein-protein interaction, especially the amino acid R40, since we observed conformational flexibility in this region allowing it to interact with interface residues. We consider that this analysis opens the possibility to validate experimentally the amino acids involved in protein-protein interactions and explore other regulatory complexes where chaperones are involved.

Interactions between the AraC/XylS-like transcriptional activator InvF of Salmonella Typhimurium, the RNA polymerase alpha subunit and the chaperone SicA.

Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.

Borrelia turicatae from Ticks in Peridomestic Setting, Camayeca, Mexico.

We conducted surveillance studies in Sinaloa, Mexico, to determine the circulation of tick-borne relapsing fever spirochetes. We collected argasid ticks from a home in the village of Camayeca and isolated spirochetes. Genomic analysis indicated that Borrelia turicatae infection is a threat to those living in resource-limited settings.

New records of Ornithodoros turicata (Ixodida: Argasidae) in rural and urban sites in the Mexican states of Aguascalientes and Zacatecas indicate the potential for tick-borne relapsing fever.

Soft ticks from the Ornithodoros genus are vectors of relapsing fever (RF) spirochetes around the world. In Mexico, they were originally described in the 19th century. However, few recent surveillance studies have been conducted in Mexico, and regions where RF spirochetes circulate remain vague. Here, the presence of soft ticks in populated areas was assessed in two sites from the Mexican states of Aguascalientes and Zacatecas. Argasidae ticks were collected, identified by morphology and mitochondrial 16S rDNA gene sequencing, and tested for RF borreliae. The specimens in both sites were identified as Ornithodoros turicata but no RF spirochetes were detected. These findings emphasize the need to update the distribution of these ticks in multiple regions of Mexico and to determine the circulation of RF borreliosis in humans and domestic animals.

Detection of Rickettsia amblyommatis and Rickettsia bellii in ticks collected from pet dogs in peri-urban and rural areas in Yucatan, Mexico.

Rickettsia species are bacteria that may cause multiple diseases in animals and humans, via transmission through multiple arthropod vectors. Routine surveillance of Rickettsia spp. within vectors is critical to determine their presence and risk to mammalian hosts within human populations. Therefore, to better characterize the circulating Rickettsia species in an understudied region we targeted pet dogs to survey. Ticks were collected from pet dogs in three populations of the Yucatan where we tested for the presence of Rickettsia spp. by PCR in metagenomic DNA. In these ticks removed from pet dogs we detected Rickettsia amblyommatis and Rickettsia bellii in Amblyomma auriculatum, Amblyomma ovale and Amblyomma mixtum ticks obtained in a rural community in the Mexican state of Yucatan. This is the first report detecting both species for this state in Mexico, underpinning the importance of more routine surveillance.

Case Report: Exposure to Relapsing Fever Group Borreliae in Patients with Undifferentiated Febrile Illness in Mexico.

Relapsing fever (RF) borreliosis is a neglected disease in Mexico. A retrospective serological survey using diagnostic antigens GlpQ and BipA from Borrelia turicatae was performed to evaluate human exposure to RF borreliae. Seventy serum samples were used from a cohort of patients with undifferentiated febrile illness in Mexico. Four samples were positive to GlpQ and three to BipA. Results indicate that RF borreliae continue to circulate in regions of Mexico and pose a risk to human health.

Distribution of CRISPR-Cas systems in the Burkholderiaceae family and its biological implications.

CRISPR-Cas systems are composed of repeated sequences separated by non-repeated sequences that are near genes coding for Cas proteins, which are involved in the function of these systems. Their function has been mostly related to "genetic immunity" against foreign genetic material, among other roles. Interest in them increased after their use in genetic manipulation was uncovered and surveys to find and classify them have been done in several bacterial groups. To determine the presence of these genetic elements in the Burkholderiaceae family members, a bioinformatic approach was followed. Attention in this family comes as it is formed by a great diversity of microorganisms that include opportunistic and true pathogens, and symbiotic and saprophytic organisms, among others. Results show that, in contrast to other bacterial groups, only 8.4% of family members harbor complete CRISPR-Cas systems and the rest either do not have one or have remains or sections of one. Analyses of the spacer sequences indicated that most of them have identity to sections of the same genomes they were found, while a few had identities with either plasmids or phages. The genus with the higher proportion of self-directed spacers is Ralstonia, and their possible roles are discussed. Most of the systems (60%) belong to the class I subtype I-E and a few to subtypes I-C (13.3%), I-F (18.3%), II-C (5%), IV-A (1.7%) and V-C (1.7%). To the best of our knowledge, this is the first study to uncover the CRISPR-Cas system for the whole Burkholderiaceae family.

Burkholderia orbicola sp. nov., a novel species within the Burkholderia cepacia complex.

Genome analysis of strains placed in the NCBI genome database as Burkholderia cenocepacia defined nine genomic species groups. The largest group (259 strains) corresponds to B. cenocepacia and the second largest group (58 strains) was identified as "Burkholderia servocepacia", a Burkholderia species classification which has not been validly published. The publication of "B. servocepacia" did not comply with Rule 27 and Recommendation 30 from the International Code of Nomenclature of Prokaryotes (ICNP) and have errors in the type strain name and the protologue describing the novel species. Here, we correct the position of this species by showing essential information that meets the criteria defined by ICNP. After additional analysis complying with taxonomic criteria, we propose that the invalid "B. servocepacia" be renamed as Burkholderia orbicola sp. nov. The original study proposing "B. servocepacia" was misleading, because this name derives from the Latin "servo" meaning "to protect/watch over", and the authors proposed this based on the beneficial biocontrol properties of several strains within the group. However, it is clear that "B. servocepacia" isolates are capable of opportunistic infection, and the proposed name Burkholderia orbicola sp. nov. takes into account these diverse phenotypic traits. The type strain is TAtl-371 T (= LMG 30279 T = CM-CNRG 715 T).

Long-chain fatty acids alter transcription of Helicobacter pylori virulence and regulatory genes.

Infection with Helicobacter pylori is one of the most important risk factors for developing gastric cancer (GC). The type IV secretion system (T4SS) encoded in the cag pathogenicity island is the main virulence factor of H. pylori associated with GC. Additionally, other virulence factors have been shown to play a role in the H. pylori virulence, such as vacuolizing cytotoxin (VacA), urease, flagella, and adhesins. Long-chain fatty acids (LCFAs) are signaling molecules that affect the transcription of virulence genes in several pathogenic bacteria such as Salmonella enterica, Vibrio cholerae, Pseudomonas aeruginosa and Mycobacterium tuberculosis. However, the effect of LCFAs on the transcription of H. pylori virulence and regulatory genes remains unknown. Here we analyzed whether the transcription of virulence genes that encode T4SS and cellular envelope components, flagellins, adhesins, toxins, urease, as well as the transcription of different regulatory genes of the H. pylori strain 26695, are altered by the presence of five distinct LCFAs: palmitic, stearic, oleic, linoleic, and linolenic acids. Palmitic and oleic acids up-regulated the transcription of most of the virulence genes tested, including cagL, cagM, flaB, sabA, mraY and vacA, as well as that of the genes encoding the transcriptional regulators NikR, Fur, CheY, ArsR, FlgR, HspR, HsrA, Hup, and CrdR. In contrast, the other LCFAs differentially affected the transcription of the virulence and regulatory genes assessed. Our data show that LCFAs can act as signaling molecules that control the transcription of the H. pylori virulome.

Point mutations in Candida glabrata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (CgHMGR) decrease enzymatic activity and substrate/inhibitor affinity.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters KM and Vmax, the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR.

Burkholderia species in human infections in Mexico: Identification of B. cepacia, B. contaminans, B. multivorans, B. vietnamiensis,B. pseudomallei and a new Burkholderia species.

BACKGROUND: Burkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described. METHODOLOGY/PRINCIPAL FINDINGS: In this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia. CONCLUSIONS/SIGNIFICANCE: The presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.

The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit.

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

An update of the unceasingly growing and diverse AraC/XylS family of transcriptional activators.

Transcriptional factors play an important role in gene regulation in all organisms, especially in Bacteria. Here special emphasis is placed in the AraC/XylS family of transcriptional regulators. This is one of the most abundant as many predicted members have been identified and more members are added because more bacterial genomes are sequenced. Given the way more experimental evidence has mounded in the past decades, we decided to update the information about this captivating family of proteins. Using bioinformatics tools on all the data available for experimentally characterized members of this family, we found that many members that display a similar functional classification can be clustered together and in some cases they have a similar regulatory scheme. A proposal for grouping these proteins is also discussed. Additionally, an analysis of surveyed proteins in bacterial genomes is presented. Altogether, the current review presents a panoramic view into this family and we hope it helps to stimulate future research in the field.

Description of two fatal cases of melioidosis in Mexican children with acute pneumonia: case report.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In Mexico, the disease is rarely diagnosed in humans and there is no evidence of simultaneous environmental isolation of the pathogen. Here, we describe clinical profiles of fatal cases of melioidosis in two children, in a region without history of that disease. CASE PRESENTATION: About 48 h before onset of symptoms, patients swam in a natural body of water, and thereafter they rapidly developed fatal septicemic illness. Upon necropsy, samples from liver, spleen, lung, cerebrospinal fluid, and bronchial aspirate tissues contained Burkholderia pseudomallei. Environmental samples collected from the locations where the children swam also contained B. pseudomallei. All the clinical and environmental strains showed the same BOX-PCR pattern, suggesting that infection originated from the area where the patients were swimming. CONCLUSIONS: The identification of B. pseudomallei confirmed that melioidosis disease exists in Sonora, Mexico. The presence of B. pseudomallei in the environment may suggest endemicity of the pathogen in the region. This study highlights the importance of strengthening laboratory capacity to prevent and control future melioidosis cases.

The Salmonella Typhimurium InvF-SicA complex is necessary for the transcription of sopB in the absence of the repressor H-NS.

Expression of virulence factors in non-typhoidal Salmonella enterica depends on a wide variety of general and specific transcriptional factors that act in response to multiple environmental signals. Expression of genes for cellular invasion located in the Salmonella pathogenicity island 1 (SPI-1) is tightly regulated by several transcriptional regulators arrayed in a cascade, while repression of this system is exerted mainly by H-NS. In SPI-1, H-NS represses the expression mainly by binding to the regulatory region of hilA and derepression is exercised mainly by HilD. However, the possible regulatory role of H-NS in genes downstream from HilD and HilA, such as those regulated by InvF, has not been fully explored. Here the role of H-NS on the expression of sopB, an InvF dependent gene encoded in SPI-5, was evaluated. Our data show that InvF is required for the expression of sopB even in the absence of H-NS. Furthermore, in agreement with previous results on other InvF-regulated genes, we found that the expression of sopB requires the InvF/SicA complex. Our results support that SicA is not required for DNA binding nor for increasing affinity of InvF to DNA in vitro. Moreover, by using a bacterial two-hybrid system we were able to identify interactions between SicA and InvF. Lastly, protein-protein interaction assays suggest that InvF functions as a monomer. Derived from these results we postulate that the InvF/SicA complex does not act on sopB as an anti-H-NS factor; instead, it seems to induce the expression of sopB by acting as a classical transcriptional regulator.

Paraburkholderia lycopersici sp. nov., a nitrogen-fixing species isolated from rhizoplane of Lycopersicon esculentum Mill. var. Saladette in Mexico.

A survey of our in-house bacterial collection identified a group of six strains isolated from the tomato rhizoplane that possessed 16S rRNA gene sequences with 98.2% sequence similarity to Paraburkholderia pallida, suggesting that these strains represented a novel species. Multilocus sequence analysis using gltB, lepA and recA gene sequences showed the clustering of the strains and the BOX-PCR patterns were similar among these strains. The average nucleotide identity and the DNA-DNA virtual hybridization of strain TNe-862T was <89% and <34%, respectively, to the genomes of any sequenced Paraburkholderia species. The genome of strain TNe-862T possessed all the genes necessary for nitrogen fixation and biosynthesis of indoleacetic acid and antimicrobials terpenes, phosphonates and bacteriocins. It also contained genes for metal resistance, xenobiotic degradation, and hydrolytic enzymes such as a putative chitinase and isoamylase. Even though the strain contained potential genes for degradation of cellulose and starch, the bacterium was unable to utilize these substrates in culture medium. The genome encoded flagella and pili as well as multiple chemotaxis systems. In addition, genes encoding for the type I, II, IV, V and VI secretion systems were also present. The strains grow up to 42°C and 5% NaCl. The optimum growth pH was 8. The major cellular fatty acids were C16:0 and C18:1 ω7c. Based on this polyphasic analysis, these strains represent a novel species in the genus Paraburkholderia, for which the name Paraburkholderia lycopersici sp. nov. is proposed. The type strain is TNe-862T (=LMG 26415T=CIP 110323T).

Draft genome of five Cupriavidus plantarum strains: agave, maize and sorghum plant-associated bacteria with resistance to metals.

Five strains of Cupriavidus plantarum, a metal-resistant, plant-associated bacterium, were selected for genome sequencing through the Genomic Encyclopedia of Bacteria and Archaea (GEBA) Phase IV project at the Joint Genome Institute (JGI) of the U.S. Department of Energy (DOE). The genome of the strains was in the size range of 6.2-6.4 Mbp and encoded 5605-5834 proteins; 16.9-23.7% of these genes could not be assigned to a COG-associated functional category. The G + C content was 65.83-65.99%, and the genomes encoded 59-67 stable RNAs. The strains were resistant in vitro to arsenite, arsenate, cobalt, chromium, copper, nickel and zinc, and their genomes possessed the resistance genes for these metals. The genomes also encoded the biosynthesis of potential antimicrobial compounds, such as terpenes, phosphonates, bacteriocins, betalactones, nonribosomal peptides, phenazine and siderophores, as well as the biosynthesis of cellulose and enzymes such as chitinase and trehalase. The average nucleotide identity (ANI) and DNA-DNA in silico hybridization of the genomes confirmed that C. plantarum is a single species. Moreover, the strains cluster within a single group upon multilocus sequence analyses with eight genes and a phylogenomic analyses. Noteworthy, the ability of the species to tolerate high concentrations of different metals might prove useful for bioremediation of naturally contaminated environments.

Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: A proposal for biocontrol of phytopathogenic fungi.

Biocontrol has emerged in recent years as an alternative to pesticides. Given the importance of environmental preservation using biocontrol, in this study two antagonistic bacteria against phytopathogenic fungi were isolated and evaluated. These bacterial strains, identified as Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24, inhibited (70 to 80%) the development of two phytopathogens of economic importance: the fungus Rhizoctonia solani RhCh-14, isolated from chili pepper, and the oomycete Pythium ultimum PyFr-14, isolated from tomato. The spectrum was not limited to the previous pathogens, but also to other phytopathogenic fungus, some bacteria and other oomycetes. Fungi-bacteria microcultures observed with optical and scanning electron microscopy revealed hyphae disintegration and pores formation. The antifungal activity was found also in the supernatant, suggesting a diffusible compound is present. Innocuous tests on tobacco leaves, blood agar, bean seed germination and in Galleria mellonella larvae showed that strain NMA1017 has the potential to be a biocontrol agent. Greenhouse experiments with bean plants inoculated with P. polymyxa exhibited the efficacy to inhibit the growth of R. solani and P. ultimum. Furthermore, P. polymyxa NMA1017 showed plant growth promotion activities, such as siderophore synthesis and nitrogen fixation which can contribute to the crop development.