An Extracytoplasmic Function Sigma Factor Required for Full Virulence in Xanthomonas citri pv. citri.

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.

Environmental Factors Modulate the Role of orf21 Sigma Factor in Clavulanic Acid Production in Streptomyces Clavuligerus ATCC27064.

Sigma factors and sigma factor-related mechanisms control antibiotic production in Streptomyces. In this contribution, the orf21 gene was overexpressed in the wild-type strain of Streptomyces clavuligerus ATCC2764, yielding S. clavuligerus/pIORF21, to further evaluate its regulatory effect on clavulanic acid (CA) biosynthesis under different culture medium conditions. The orf21 overexpression, regulated under the constitutive promoter ermE*, led to 2.6-fold increase in CA production in GSPG medium, and a 1.8-fold decrease using ISP medium. As for GYM and MYM media, S. clavuligerus/pIORF21 strain showed higher aerial mycelium production compared to control. Glycerol uptake rate profile was affected by orf21 overexpression. Furthermore, in GSPG, S. clavuligerus/pIORF21 slightly increased the expression of adpA and gcas genes, whilst, in ISP, the claR gene expression was drastically reduced, which is consistent with a decreased CA production, observed in this medium. These findings suggest the protein encoded by the orf21 gene plays a role in the regulation of CA biosynthesis as a response to the nutritional composition of the medium.

The Stress-Responsive Alternative Sigma Factor SigB of Bacillus subtilis and Its Relatives: An Old Friend With New Functions.

Alternative sigma factors have led the core RNA polymerase (RNAP) to recognize different sets of promoters to those recognized by the housekeeping sigma A-directed RNAP. This change in RNAP promoter selectivity allows a rapid and flexible reformulation of the genetic program to face environmental and metabolic stimuli that could compromise bacterial fitness. The model bacterium Bacillus subtilis constitutes a matchless living system in the study of the role of alternative sigma factors in gene regulation and physiology. SigB from B. subtilis was the first alternative sigma factor described in bacteria. Studies of SigB during the last 40 years have shown that it controls a genetic universe of more than 150 genes playing crucial roles in stress response, adaption, and survival. Activation of SigB relies on three separate pathways that specifically respond to energy, environmental, and low temperature stresses. SigB homologs, present in other Gram-positive bacteria, also play important roles in virulence against mammals. Interestingly, during recent years, other unexpected B. subtilis responses were found to be controlled by SigB. In particular, SigB controls the efficiencies of spore and biofilm formation, two important features that play critical roles in adaptation and survival in planktonic and sessile B. subtilis communities. In B. subtilis, SigB induces the expression of the Spo0E aspartyl-phosphatase, which is responsible for the blockage of sporulation initiation. The upregulated activity of Spo0E connects the two predominant adaptive pathways (i.e., sporulation and stress response) present in B. subtilis. In addition, the RsbP serine-phosphatase, belonging to the energy stress arm of the SigB regulatory cascade, controls the expression of the key transcription factor SinR to decide whether cells residing in the biofilm remain in and maintain biofilm growth or scape to colonize new niches through biofilm dispersal. SigB also intervenes in the recognition of and response to surrounding microorganisms, a new SigB role that could have an agronomic impact. SigB is induced when B. subtilis is confronted with phytopathogenic fungi (e.g., Fusarium verticillioides) and halts fungal growth to the benefit of plant growth. In this article, we update and review literature on the different regulatory networks that control the activation of SigB and the new roles that have been described the recent years.

Evolution of a Sigma Factor: An All-In-One of Gene Duplication, Horizontal Gene Transfer, Purifying Selection, and Promoter Differentiation.

Sigma factors are an essential part of bacterial gene regulation and have been extensively studied as far as their molecular mechanisms and protein structure are concerned. However, their molecular evolution, especially for the alternative sigma factors, is poorly understood. Here, we analyze the evolutionary forces that have shaped the rpoH sigma factors within the alphaproteobacteria. We found that an ancient duplication gave rise to two major groups of rpoH sigma factors and that after this event horizontal gene transfer (HGT) occurred in rpoH 1 group. We also noted that purifying selection has differentially affected distinct parts of the gene; singularly, the gene segment that encodes the region 4.2, which interacts with the -35 motif of the RpoH-dependent genes, has been under relaxed purifying selection. Furthermore, these two major groups are clearly differentiated from one another regarding their promoter selectivity, as rpoH 1 is under the transcriptional control of σ(70) and σ(32), whereas rpoH 2 is under the transcriptional control of σ(24). Our results suggest a scenario in which HGT, gene loss, variable purifying selection and clear promoter specialization occurred after the ancestral duplication event. More generally, our study offers insights into the molecular evolution of alternative sigma factors and highlights the importance of analyzing not only the coding regions but also the promoter regions.

Caracterização da superexpressão do fator sigma ECF σx em Pseudomonas aeruginosa PA14 / Characterization ofthe ECF sigma fator σx overexpression in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando σx (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem σx à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de σx. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de σ70, ao contrário dos relatos na literatura da dependência da região de ligação a σx. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de σx, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando σx apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa

Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa